elife02450.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN"  "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">02450</article-id><article-id pub-id-type="doi">10.7554/eLife.02450</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biochemistry</subject></subj-group><subj-group subj-group-type="heading"><subject>Microbiology and infectious disease</subject></subj-group></article-categories><title-group><article-title>GE23077 binds to the RNA polymerase ‘i’ and ‘i+1’ sites and prevents the binding of initiating nucleotides</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-11087"><name><surname>Zhang</surname><given-names>Yu</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/><xref ref-type="other" rid="dataro5"/><xref ref-type="other" rid="dataro6"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-11085"><name><surname>Degen</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf3"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-11096"><name><surname>Ho</surname><given-names>Mary X</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf8"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/><xref ref-type="other" rid="dataro5"/><xref ref-type="other" rid="dataro6"/></contrib><contrib contrib-type="author" id="author-11097"><name><surname>Sineva</surname><given-names>Elena</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" id="author-11088"><name><surname>Ebright</surname><given-names>Katherine Y</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf5"/></contrib><contrib contrib-type="author" id="author-11089"><name><surname>Ebright</surname><given-names>Yon W</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-11098"><name><surname>Mekler</surname><given-names>Vladimir</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" id="author-12303"><name><surname>Vahedian-Movahed</surname><given-names>Hanif</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" id="author-11086"><name><surname>Feng</surname><given-names>Yu</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" id="author-11100"><name><surname>Yin</surname><given-names>Ruiheng</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" id="author-11101"><name><surname>Tuske</surname><given-names>Steve</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" id="author-11102"><name><surname>Irschik</surname><given-names>Herbert</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" id="author-11103"><name><surname>Jansen</surname><given-names>Rolf</given-names></name><xref ref-type="aff" rid="aff5"/><xref ref-type="fn" rid="con13"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" id="author-11104"><name><surname>Maffioli</surname><given-names>Sonia</given-names></name><xref ref-type="aff" rid="aff6"/><xref ref-type="fn" rid="con14"/><xref ref-type="fn" rid="conf6"/></contrib><contrib contrib-type="author" id="author-11105"><name><surname>Donadio</surname><given-names>Stefano</given-names></name><xref ref-type="aff" rid="aff6"/><xref ref-type="fn" rid="con15"/><xref ref-type="fn" rid="conf7"/></contrib><contrib contrib-type="author" id="author-11094"><name><surname>Arnold</surname><given-names>Eddy</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con16"/><xref ref-type="fn" rid="conf8"/></contrib><contrib contrib-type="author" corresp="yes" id="author-11021"><name><surname>Ebright</surname><given-names>Richard H</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con17"/><xref ref-type="fn" rid="conf4"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/><xref ref-type="other" rid="dataro3"/><xref ref-type="other" rid="dataro4"/><xref ref-type="other" rid="dataro5"/><xref ref-type="other" rid="dataro6"/></contrib><aff id="aff1"><institution content-type="dept">Waksman Institute</institution>, <institution>Rutgers University</institution>, <addr-line><named-content content-type="city">Piscataway</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Chemistry and Chemical Biology</institution>, <institution>Rutgers University</institution>, <addr-line><named-content content-type="city">Piscataway</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Center for Advanced Biotechnology and Medicine</institution>, <institution>Rutgers University</institution>, <addr-line><named-content content-type="city">Piscataway</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution content-type="dept">Natural Products Chemistry</institution>, <institution>Helmholtz Centre for Infection Research</institution>, <addr-line><named-content content-type="city">Braunschweig</named-content></addr-line>, <country>Germany</country></aff><aff id="aff5"><institution content-type="dept">Microbial Drugs</institution>, <institution>Helmholtz Centre for Infection Research</institution>, <addr-line><named-content content-type="city">Braunschweig</named-content></addr-line>, <country>Germany</country></aff><aff id="aff6"><institution>Naicons Srl</institution>, <addr-line><named-content content-type="city">Milan</named-content></addr-line>, <country>Italy</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Storz</surname><given-names>Gisela</given-names></name><role>Reviewing editor</role><aff><institution>National Institute of Child Health and Human Development</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>ebright@waksman.rutgers.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>22</day><month>04</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02450</elocation-id><history><date date-type="received"><day>02</day><month>02</month><year>2014</year></date><date date-type="accepted"><day>11</day><month>03</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Zhang et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Zhang et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife02450.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="commentary" xlink:href="10.7554/eLife.02840"/><related-article ext-link-type="doi" id="ra2" related-article-type="article-reference" xlink:href="10.7554/eLife.02451"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02450.001</object-id><p>Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center ‘i’ and ‘i+1’ nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.001">http://dx.doi.org/10.7554/eLife.02450.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02450.002</object-id><title>eLife digest</title><p>As increasing numbers of bacteria become resistant to antibiotics, new drugs are needed to fight bacterial infections. To develop new antibacterial drugs, researchers need to understand how existing antibiotics work. There are many ways to kill bacteria, but one of the most effective is to target an enzyme called bacterial RNA polymerase. If bacterial RNA polymerase is prevented from working, bacteria cannot synthesize RNA and cannot survive.</p><p>GE23077 (GE for short) is an antibiotic produced by bacteria found in soil. Although GE stops bacterial RNA polymerase from working, and thereby kills bacteria, it does not affect mammalian RNA polymerases, and so does not kill mammalian cells. Understanding how GE works could help with the development of new antibacterial drugs.</p><p>Zhang et al. present results gathered from a range of techniques to show how GE inhibits bacterial RNA polymerase. These show that GE works by binding to a site on RNA polymerase that is different from the binding sites of previously characterized antibacterial drugs. The mechanism used to inhibit the RNA polymerase is also different.</p><p>The newly identified binding site has several features that make it an unusually attractive target for development of antibacterial compounds. Bacteria can become resistant to an antibiotic if genetic mutations lead to changes in the site the antibiotic binds to. However, the site that GE binds to on RNA polymerase is essential for RNA polymerase to function and so cannot readily be changed without crippling the enzyme. Therefore, this type of antibiotic resistance is less likely to develop.</p><p>In addition, the newly identified binding site for GE on RNA polymerase is located next to the binding site for a current antibacterial drug, rifampin. Zhang et al. therefore linked GE and rifampin to form a two-part (‘bipartite’) compound designed to bind simultaneously to the GE and the rifampin binding sites. This compound was able to inhibit drug-resistant RNA polymerases tens to thousands of times more potently than GE or rifampin alone.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.002">http://dx.doi.org/10.7554/eLife.02450.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>RNA polymerase</kwd><kwd>RNA polymerase-promoter open complex</kwd><kwd>transcription</kwd><kwd>transcription initiation</kwd><kwd>inhibitor</kwd><kwd>bipartite inhibitor</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organisms</title><kwd><italic>E. coli</italic></kwd><kwd><italic>Streptococcus pyogenes</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>GM041376</award-id><principal-award-recipient><name><surname>Ebright</surname><given-names>Richard H</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AI104660</award-id><principal-award-recipient><name><surname>Ebright</surname><given-names>Richard H</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000011</institution-id><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Ebright</surname><given-names>Richard H</given-names></name></principal-award-recipient></award-group><funding-statement>The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The cyclic-peptide antibiotic GE23077 inhibits bacterial RNA polymerase through a novel target that exhibits low susceptibility to target-based resistance and that enables synthesis of bipartite inhibitors that are exceptionally potent and refractory to target-based resistance.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>GE23077 (GE) is a cyclic-peptide antibiotic produced by the soil bacterium <italic>Actinomadura</italic> sp. DSMZ 13491 (<xref ref-type="fig" rid="fig1">Figure 1A</xref>; <xref ref-type="bibr" rid="bib10">Ciciliato et al., 2004</xref>). GE exhibits antibacterial activity against both Gram-negative and Gram-positive bacterial pathogens in culture, including <italic>Moraxella catarrhalis</italic> and <italic>Streptococcus pyogenes</italic> (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1A</xref>; <xref ref-type="bibr" rid="bib10">Ciciliato et al., 2004</xref>). GE inhibits both Gram-negative and Gram-positive bacterial RNA polymerase (RNAP) in vitro, but does not inhibit human RNAP I, II, or III in vitro (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1B</xref>; <xref ref-type="bibr" rid="bib10">Ciciliato et al., 2004</xref>). Analysis of the kinetics of inhibition suggests that GE inhibits RNAP at a stage subsequent to the formation of the RNAP-template complex (<xref ref-type="bibr" rid="bib58">Sarubbi et al., 2004</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02450.003</object-id><label>Figure 1.</label><caption><title>Mechanism of transcription inhibition by GE: inhibition of first nucleotide addition in transcription initiation.</title><p>(<bold>A</bold>) Structure of GE. dmaDap, N<sup>β</sup>-(Z-2,3-dimethylacryloyl)-α,β-diaminopropionic acid; dhGln, β,γ-dihydroxy-glutamine; Ama, aminomalonic acid; aThr, allothreonine; iSer, isoserine. Wavy bonds, previously undefined stereochemistry. (<bold>B</bold>) GE does not inhibit formation of a transcription initiation complex. (<bold>C</bold>) GE inhibits nucleotide addition in transcription initiation (primer-dependent transcription initiation). (<bold>D</bold>) GE does not inhibit nucleotide addition in transcription elongation (elongation from halted TEC containing 29 nt RNA product). See <xref ref-type="fig" rid="fig1s1 fig1s2">Figure 1—figure supplements 1, 2</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.003">http://dx.doi.org/10.7554/eLife.02450.003</ext-link></p></caption><graphic xlink:href="elife02450f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>GE inhibits nucleotide addition in transcription initiation (<italic>de novo</italic> transcription initiation).</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.004">http://dx.doi.org/10.7554/eLife.02450.004</ext-link></p></caption><graphic xlink:href="elife02450fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>GE does not inhibit nucleotide addition in transcription elongation (reconstituted transcription elongation complexes).</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.005">http://dx.doi.org/10.7554/eLife.02450.005</ext-link></p></caption><graphic xlink:href="elife02450fs002"/></fig></fig-group></p><p>GE is a non-ribosomally-synthesized cyclic heptapeptide (<xref ref-type="fig" rid="fig1">Figure 1A</xref>; <xref ref-type="bibr" rid="bib40">Marazzi et al., 2005</xref>). The stereochemistry at four chiral centers of GE has been defined based on acid hydrolysis and gas chromatography, but the stereochemistry at five other chiral centers has not been defined (<xref ref-type="fig" rid="fig1">Figure 1A</xref>; <xref ref-type="bibr" rid="bib40">Marazzi et al., 2005</xref>). Analogs of GE having modifications of the dmaDap, dhGln, and Ama residues, have been prepared by semi-synthetic derivatization of GE (<xref ref-type="bibr" rid="bib42">Mariani et al., 2005</xref>).</p><p>Here we report the target and mechanism of transcription inhibition by GE. In addition, we report a series of crystal structures—including the first crystal structure of a substrate complex for de novo transcription initiation by a multisubunit RNAP—that define the structural relationships between GE and RNAP, GE and promoter DNA, GE and NTPs, and GE and rifamycins.</p><p>Our results show that GE inhibits RNAP through a novel binding site and novel mechanism. GE inhibits RNAP by binding to a site—the ‘GE target’—that overlaps the RNAP active-center ‘i’ and ‘i+1’ sites and that includes coordinating ligands of the RNAP active-center catalytic Mg<sup>2+</sup> ion, Mg<sup>2+</sup>(I). Binding of GE sterically precludes binding of initiating NTPs to the i site, i+1 site, and Mg<sup>2+</sup>(I), and thereby blocks transcription initiation. GE is the first identified example of a non-nucleoside RNAP inhibitor that functions through direct interaction with the core catalytic components of the RNAP active-center: the i site, i+1 site, and Mg<sup>2+</sup>(I).</p><p>Our results further show that the GE target has three features that make it an unusually attractive target—a ‘privileged target’—for antibacterial drug discovery involving RNAP. First, the GE target includes functionally critical residues of the RNAP active center that cannot be substituted without loss of RNAP activity, and, therefore, that cannot be substituted to yield resistant mutants. Accordingly, the target-based resistance spectrum for GE is unusually small. Second, the GE target does not overlap the rifamycin target (the target of the most important RNAP inhibitors in current clinical use in antibacterial therapy; <xref ref-type="bibr" rid="bib27">Ho et al., 2009</xref>). Accordingly, GE exhibits no or negligible cross-resistance with rifamycins. Third, the GE target is immediately adjacent to the rifamycin target. Accordingly, it is possible to link GE to a rifamycin to construct a bipartite inhibitor that binds simultaneously to the GE target and the rifamycin target and, therefore, that is exceptionally potent and exceptionally refractory to target-based resistance.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Mechanism of inhibition by GE: inhibition of first nucleotide addition in transcription initiation</title><p>To define the mechanism of transcription inhibition by GE, we assessed effects of GE on individual reaction steps in transcription initiation and transcription elongation. <xref ref-type="fig" rid="fig1">Figure 1B</xref> shows that GE does not inhibit steps in transcription initiation up to and including formation of a competitor-resistant RNAP-promoter open complex (RP<sub>o</sub>). We infer that GE does not inhibit promoter binding, loading of promoter DNA into the RNAP active-center cleft, or promoter unwinding.</p><p>The results in <xref ref-type="fig" rid="fig1">Figure 1C</xref> show that GE inhibits nucleotide addition in transcription initiation. GE inhibits both primer-dependent transcription initiation (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), and de novo transcription initiation (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). In primer-dependent transcription initiation, GE inhibits the first nucleotide-addition step, inhibiting the synthesis of a 3-nt RNA product from a 2-nt RNA primer and an NTP. In de novo transcription initiation, GE inhibits the first nucleotide-addition step, inhibiting the synthesis of a 2-nt RNA product from initiating NTPs.</p><p>The results in <xref ref-type="fig" rid="fig1">Figure 1D</xref> show that GE does not inhibit nucleotide addition in transcription elongation. GE does not inhibit transcription elongation upon addition of NTPs to a halted elongation complex (<xref ref-type="fig" rid="fig1">Figure 1D</xref>), and GE does not inhibit single nucleotide addition upon addition of an NTP to an elongation complex reconstituted from RNAP and a synthetic nucleic acid scaffold (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>).</p><p>We conclude that GE specifically inhibits nucleotide addition in transcription initiation. The observation that GE inhibits nucleotide addition in initiation but not in elongation suggests that GE functions through a binding site that is available in RP<sub>o</sub> but that is not available in an elongation complex—for example, a binding site that overlaps the RNAP active-center i and i+1 nucleotide binding sites, or the path of the RNA product from the i and i+1 nucleotide binding sites, and that therefore would be unoccupied in RP<sub>o</sub> but occupied by RNA in an elongation complex.</p><p>The mechanism of transcription inhibition of GE is reminiscent of, but differs from, the mechanism of transcription inhibition by rifampin (Rif) and other members of the rifamycin class. Like GE, Rif does not inhibit formation of RP<sub>o</sub> (<xref ref-type="fig" rid="fig1">Figure 1B</xref>; <xref ref-type="bibr" rid="bib44">McClure and Cech, 1978</xref>). Also like GE, Rif inhibits nucleotide addition in transcription initiation, but does not inhibit nucleotide addition in transcription elongation (<xref ref-type="fig" rid="fig1">Figure 1C,D</xref>; <xref ref-type="bibr" rid="bib68">Sippel and Hartmann, 1968</xref>). However, in contrast to GE, Rif does not generally inhibit the first nucleotide-addition step in transcription initiation (<xref ref-type="fig" rid="fig1">Figure 1C</xref>; <xref ref-type="bibr" rid="bib44">McClure and Cech, 1978</xref>). Rif generally only inhibits synthesis of &gt;2–3-nt RNA products and does so by binding to a site along the path of RNA from the RNAP active-center and sterically blocking RNA extension (<xref ref-type="bibr" rid="bib6">Campbell et al., 2001</xref>; <xref ref-type="bibr" rid="bib20">Feklistov et al., 2008</xref>). The observation that GE inhibits synthesis of 2-nt RNA products, whereas Rif generally only inhibits synthesis of &gt;2–3-nt RNA products, suggests that GE functions through a binding site located closer than the Rif binding site to the RNAP active-center.</p><p>The mechanism of transcription inhibition by GE also differs from the mechanisms of transcription inhibition by other previously characterized RNAP inhibitors. Sorangicin (Sor) functions through the same binding site on RNAP as Rif and inhibits synthesis only of &gt;2–3-nt RNA products (<xref ref-type="bibr" rid="bib7">Campbell et al., 2005</xref>). Myxopyronin (Myx), corallopyronin (Cor), ripostatin (Rip), and lipiarmycin (Lpm) inhibit formation of RP<sub>o</sub> (<xref ref-type="bibr" rid="bib27">Ho et al., 2009</xref>). Streptolydigin (Stl), CBR703 (CBR), and microcin J25 (MccJ25) inhibit nucleotide addition in both initiation and elongation (<xref ref-type="bibr" rid="bib2">Artsimovitch et al., 2003</xref>; <xref ref-type="bibr" rid="bib48">Mukhopadhyay et al., 2004</xref>; <xref ref-type="bibr" rid="bib27">Ho et al., 2009</xref>). We conclude that GE inhibits transcription through a novel mechanism.</p></sec><sec id="s2-2"><title>Target of inhibition by GE: RNAP active-center i and i+1 sites</title><sec id="s2-2-1"><title>Isolation and characterization of GE-resistant mutants</title><p>To identify the target in RNAP for GE, we performed saturation mutagenesis of genes encoding <italic>Escherichia coli</italic> RNAP β and β′ subunits, and isolated and characterized mutants conferring GE-resistance (GE<sup>R</sup>). We performed saturation mutagenesis using a set of ‘doped’ oligonucleotide primers designed to introduce all possible nucleotide substitutions at all codons for all residues located within 30 Å of the RNAP active-center i and i+1 sites (primer sequences in <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2A</xref>). We identified 33 independent single-substitution GE<sup>R</sup> mutants (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). All mapped to the RNAP β subunit (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). The GE<sup>R</sup> substitutions comprised six distinct substitutions at three sites in RNAP β: residues 565, 566, and 684 (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Minimal inhibitory concentration (MIC) assays indicate that all six GE<sup>R</sup> substitutions result in at least moderate resistance (≥fourfold higher MIC) and that two result in high-level resistance (≥16-fold higher MIC; <xref ref-type="fig" rid="fig2">Figure 2A</xref>; <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2B</xref>). Complementation assays indicate that each GE<sup>R</sup> mutant is able to complement an <italic>rpoB</italic><sup>ts</sup> mutant for growth at the non-permissive temperature, indicating that each GE<sup>R</sup> RNAP derivative is sufficiently functional in transcription to support viability (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). RNAP purified from GE<sup>R</sup> mutants exhibited resistance in vitro (<xref ref-type="fig" rid="fig2">Figure 2B</xref>), indicating that the GE<sup>R</sup> phenotype at the cellular level is attributable to resistance at the enzymatic level. We conclude that RNAP is the functional cellular target for GE, and that RNAP β residues 565, 566, and 684 comprise a determinant essential for transcription inhibition by GE.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02450.006</object-id><label>Figure 2.</label><caption><title>Target of transcription inhibition by GE: RNAP active-center i and i+1 sites.</title><p>(<bold>A</bold>) GE<sup>R</sup> mutants obtained following saturation mutagenesis of <italic>E. coli rpoB</italic> and <italic>rpoC</italic>. (<bold>B</bold>) GE<sup>R</sup> phenotype of RNAP derivatives purified from GE<sup>R</sup> mutants. (<bold>C</bold>) The GE target overlaps the RNAP active-center region. Structure of RNAP (gray ribbons; black circle for active-center region; violet sphere for Mg<sup>2+</sup>(I); β’ non-conserved region and σ omitted for clarity; <xref ref-type="bibr" rid="bib46">Mukhopadhyay et al., 2008</xref>), showing sites of GE-resistant substitutions (green; sequences from A and <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2C</xref>). Two orthogonal views. (<bold>D</bold>) The GE target does not overlap the Rif target. Structure of RNAP, showing sites of GE<sup>R</sup> substitutions (green; sequences from A and <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2C</xref>) and Rif<sup>R</sup> substitutions (red; <xref ref-type="bibr" rid="bib30">Jin and Gross, 1988</xref>; <xref ref-type="bibr" rid="bib63">Severinov et al., 1993</xref>). (<bold>E</bold>) GE<sup>R</sup> mutants are not cross-resistant to Rif. (<bold>F</bold>) Rif<sup>R</sup> mutants are not cross-resistant to GE. See <xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplements 1, 2</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.006">http://dx.doi.org/10.7554/eLife.02450.006</ext-link></p></caption><graphic xlink:href="elife02450f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.007</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Location of GE target in sequence of RNAP β subunit.</title><p>Sequence alignments for the β subunits of bacterial RNAP (top twenty-one sequences) and corresponding subunits of human RNAP I, RNAP II, and RNAP III (bottom three sequences), showing locations of GE<sup>R</sup> substitutions in <italic>E. coli</italic> (black rectangles; sequences from <xref ref-type="fig" rid="fig2">Figure 2A</xref>) and <italic>S. pyogenes</italic> (black and gray rectangles; sequences in <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2C</xref>). Species are as follows: <italic>E. coli</italic> (ECOLI), <italic>Haemophilus influenzae</italic> (HAEIN), <italic>Vibrio cholerae</italic> (VIBCH), <italic>Pseudomonas aeruginosa</italic> (PSEAE), <italic>Treponema pallidum</italic> (TREPA), <italic>Borrelia burgdorferi</italic> (BORBU), <italic>Xylella fastidiosa</italic> (XYLFA), <italic>Campylobacter jejuni</italic> (CAMJE), <italic>Neisseria meningitidis</italic> (NEIMA), <italic>Rickettsia prowazekii</italic> (RICPR), <italic>Chlamydia trachomatis</italic> (CHLTR), <italic>Mycoplasma pneumoniae</italic> (MYCPN), <italic>Bacillus subtilis</italic> (BACSU), <italic>Staphylococcus aureus</italic> (STAAU), <italic>Streptococcus pyogenes</italic> (STRP1), <italic>Mycobacterium tuberculosis</italic> (MYCTU), <italic>Synechocystis sp.</italic> PCC 6803 (SYNY3), <italic>Aquifex aeolicus</italic> (AQUAE), <italic>Deinococcus radiodurans</italic> (DEIRA), <italic>Thermus thermophilus</italic> (THETH), <italic>Thermus aquaticus</italic> (THEAQ), and <italic>Homo sapiens</italic> (HUMAN).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.007">http://dx.doi.org/10.7554/eLife.02450.007</ext-link></p></caption><graphic xlink:href="elife02450fs003"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.008</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Relationship between GE target and targets of other RNAP inhibitors.</title><p>The GE target does not overlap the targets of Rif, Sor, Stl, CBR703, Myx, and Lpm. Structure of bacterial RNAP (gray ribbons; violet sphere for active-center Mg<sup>2+</sup>; β' nonconserved region and σ omitted for clarity; <xref ref-type="bibr" rid="bib46">Mukhopadhyay et al., 2008</xref>), showing GE target (green; <xref ref-type="fig" rid="fig2">Figure 2C</xref>) and targets of Rif and Sor (red; <xref ref-type="bibr" rid="bib53">Ovchinnikov et al., 1981</xref>, <xref ref-type="bibr" rid="bib54">1983</xref>; <xref ref-type="bibr" rid="bib37">Lisitsyn et al., 1984</xref>; <xref ref-type="bibr" rid="bib30">Jin and Gross, 1988</xref>; <xref ref-type="bibr" rid="bib63">Severinov et al., 1993</xref>, <xref ref-type="bibr" rid="bib64">1994</xref>; <xref ref-type="bibr" rid="bib22">Garibyan et al., 2003</xref>, <xref ref-type="bibr" rid="bib7">Campbell et al., 2005</xref>; <xref ref-type="bibr" rid="bib82">Xu et al., 2005</xref>; <xref ref-type="bibr" rid="bib55">Rodriguez-Verdugo et al., 2013</xref>; ES and RHE, unpublished), Stl (yellow; <xref ref-type="bibr" rid="bib38">Lisitsyn et al., 1985</xref>; <xref ref-type="bibr" rid="bib24">Heisler et al., 1993</xref>; <xref ref-type="bibr" rid="bib61">Severinov et al., 1995</xref>; <xref ref-type="bibr" rid="bib75">Tuske et al., 2005</xref>), CBR703 (blue; <xref ref-type="bibr" rid="bib2">Artsimovitch et al., 2003</xref>; X Wang and RHE, unpublished), Myx (magenta; <xref ref-type="bibr" rid="bib46">Mukhopadhyay et al., 2008</xref>), and Lpm (cyan; <xref ref-type="bibr" rid="bib15">Ebright, 2005</xref>; <xref ref-type="bibr" rid="bib70">Srivastava et al., 2011</xref>; RY Ebright, DD, and RHE, unpublished). Views as in <xref ref-type="fig" rid="fig2">Figure 2C</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.008">http://dx.doi.org/10.7554/eLife.02450.008</ext-link></p></caption><graphic xlink:href="elife02450fs004"/></fig></fig-group></p><p>Analysis of a panel of <italic>Streptococcus pyogenes</italic> mutants carrying single-substitutions within the RNAP active-center region indicates that substitutions at residues corresponding to <italic>E. coli</italic> RNAP β residues 565, 681, and 684 confer a GE<sup>R</sup> phenotype (<xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2C</xref>). We conclude that the region comprising RNAP β residues 565-566 and 681-684 constitutes a determinant essential for transcription inhibition by GE in both Gram-negative and Gram-positive bacterial RNAP.</p><p>The sites of GE<sup>R</sup> substitutions are conserved in RNAP from both Gram-negative and Gram-positive bacteria (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). This is consistent with, and accounts for, the observation that GE inhibits RNAP from both Gram-negative and Gram-positive bacteria (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1B</xref>). Two sites of GE<sup>R</sup> substitutions, β residues 681 and 684, are not conserved in human RNAP I, II, and III (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). This is consistent with, and accounts for, the observation that GE does not inhibit human RNAP I, II, and III (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1B</xref>; <xref ref-type="bibr" rid="bib10">Ciciliato et al., 2004</xref>).</p></sec><sec id="s2-2-2"><title>GE target</title><p>In the three-dimensional structure of RNAP, the sites of GE<sup>R</sup> substitutions are located adjacent to each other and form a compact determinant (‘GE target’; <xref ref-type="fig" rid="fig2">Figure 2C</xref>). The GE target is located in the RNAP active-center region (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). The GE target overlaps the RNAP active-center i and i+1 nucleotide binding sites, and comprises residues in two active-center subregions: the ‘D2 loop’ and the ‘link region’ (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). The RNAP active center contains two nucleotide binding sites—the i site and the i+1 site--flanking the catalytic Mg<sup>2+</sup> ion, Mg<sup>2+</sup>(I) (<xref ref-type="bibr" rid="bib83">Zhang and Landick, 2009</xref>). The i site serves as the binding site for the first initiating NTP in de novo transcription initiation, and as the binding site for the 3′-nucleotide of the RNA primer in primer-dependent transcription initiation and RNA product in transcription elongation. The i+1 site serves as the binding site for the second initiating NTP in de novo transcription initiation, and as the binding site for the extending NTP in primer-dependent transcription initiation and transcription elongation (<xref ref-type="bibr" rid="bib83">Zhang and Landick, 2009</xref>). The D2 loop and the link region play roles in nucleotide addition, transcriptional fidelity, and transcriptional pausing (<xref ref-type="bibr" rid="bib36">Libby et al., 1989</xref>; <xref ref-type="bibr" rid="bib35">Landick et al., 1990</xref>; <xref ref-type="bibr" rid="bib74">Toulokhonov et al., 2007</xref>; <xref ref-type="bibr" rid="bib79">Weinzierl 2010</xref>, <xref ref-type="bibr" rid="bib80">2012</xref>; <xref ref-type="bibr" rid="bib23">Gordon et al., 2012</xref>). The location of the GE target suggests that GE inhibits RNAP through direct interference with the function of the i and i+1 nucleotide binding sites and/or of Mg<sup>2+</sup>(I).</p><p>The GE target is located approximately midway between Mg<sup>2+</sup>(I) and the Rif target (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>). The location is consistent with the hypothesis of the preceding section that the inhibition of the first nucleotide-addition step by GE, but only of subsequent nucleotide-addition steps by Rif, is attributable to the closer proximity of the GE binding site to the RNAP active-center.</p></sec><sec id="s2-2-3"><title>Relationship between GE target and targets of previously characterized RNAP inhibitors</title><p>The GE target is located adjacent to, but does not overlap, the Rif target (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). Consistent with the absence of overlap, GE<sup>R</sup> mutants do not exhibit cross-resistance with Rif (<xref ref-type="fig" rid="fig2">Figure 2E</xref>; <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2D</xref>), and, conversely, Rif<sup>R</sup> mutants do not exhibit cross-resistance with GE (<xref ref-type="fig" rid="fig2">Figure 2F</xref>; <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2E</xref>; <xref ref-type="bibr" rid="bib10">Ciciliato et al., 2004</xref>).</p><p>The GE target also does not overlap the targets of the previously characterized RNAP inhibitors Sor, Myx, Cor, Rip, Lpm, Stl, and CBR703 (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). Accordingly, GE<sup>R</sup> mutants exhibit no cross-resistance with Sor, Myx, Cor, Rip, Lpm, Stl, and CBR703 (<xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2F</xref>).</p></sec><sec id="s2-2-4"><title>Unusually small size of GE target</title><p>The GE target is strikingly small. The GE target comprises only six substitutions and only three sites in <italic>E. coli</italic> RNAP (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), and has dimensions of just ∼16 Å × ∼10 Å × ∼9 Å (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). The GE target is much smaller than the Rif target (71 substitutions and 27 sites; ∼30 Å × ∼25 Å × ∼10 Å; <xref ref-type="fig" rid="fig2">Figure 2D</xref>; <xref ref-type="bibr" rid="bib30">Jin and Gross, 1988</xref>; <xref ref-type="bibr" rid="bib63">Severinov et al., 1993</xref>). The GE target also is much smaller than the targets of other RNAP inhibitors, including the Myx/Cor/Rip target (28 substitutions and 19 sites; <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>; <xref ref-type="bibr" rid="bib46">Mukhopadhyay et al., 2008</xref>), the Lpm target (30 substitutions and 20 sites; <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>; <xref ref-type="bibr" rid="bib70">Srivastava et al., 2011</xref>; DD, and RHE, unpublished), the Stl target (27 substitutions and 19 sites; <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>; <xref ref-type="bibr" rid="bib75">Tuske et al., 2005</xref>), the CBR703 target (23 substitutions and 13 sites; <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>; <xref ref-type="bibr" rid="bib2">Artsimovitch et al., 2003</xref>; X Wang and RHE, unpublished), and the MccJ25 target (86 substitutions and 52 sites; <xref ref-type="bibr" rid="bib48">Mukhopadhyay et al., 2004</xref>). The GE target also is small relative to the size of GE. We infer that the genetically defined GE target corresponds to just part of the GE binding site on RNAP, not the full GE binding site on RNAP (in contrast to the genetically defined targets of Rif and other previously characterized RNAP inhibitors, which correspond to full inhibitor binding sites; <xref ref-type="bibr" rid="bib27">Ho et al., 2009</xref>). Specifically, we infer that the GE binding site comprises not only the residues at which GE<sup>R</sup> substitutions are obtained, but also evolutionarily invariant, functionally essential, residues of the RNAP active center that cannot be substituted without loss of RNAP function, and thus cannot be substituted to confer GE-resistance. According to this hypothesis, the full GE binding site on RNAP includes not only the genetically-defined GE target, but also the full, or nearly the full, active-center i and i+1 sites; and GE bound to its target would be positioned to interfere directly, through steric clash, with function of the i and i+1 sites and/or Mg<sup>2+</sup>(I).</p><p>The unusually small size of the GE target-based resistance spectrum (six substitutions at three sites in <italic>E. coli</italic>; ∼1/10 the size of the target-based resistance spectrum for Rif, and ∼1/10 to ∼1/5 the sizes of the target-based resistance spectra for other RNAP inhibitors) has a potentially important practical implication. Namely, the frequency of spontaneous mutations yielding target-dependent GE-resistance is expected to be unusually small (∼1/10 to ∼1/5 the frequency of spontaneous mutations yielding target-dependent resistance to Rif and other RNAP inhibitors). In view of the fact that spontaneous mutations yielding target-dependent Rif-resistance are a major problem in antibacterial therapy with Rif (<xref ref-type="bibr" rid="bib27">Ho et al., 2009</xref>), the smaller size of the GE target-based resistance spectrum is a potentially important advantage.</p></sec></sec><sec id="s2-3"><title>Structural basis of inhibition by GE: crystal structure of RNAP-GE</title><sec id="s2-3-1"><title>GE binds to the GE target</title><p>To define the structural basis of transcription inhibition by GE, we determined a crystal structure of <italic>Thermus thermophilus</italic> RNAP holoenzyme in complex with GE at 3.35 Å resolution (<xref ref-type="fig" rid="fig3">Figure 3</xref>). <xref ref-type="fig" rid="fig3">Figure 3A</xref> shows that GE binds to the genetically-defined GE target, confirming the hypothesis that the GE target represents a determinant for binding of GE to RNAP. The structure shows that GE occupies the RNAP i and i+1 sites and makes direct interactions with the D2 loop, the link region, and an RNAP Asp residue and water molecule that coordinate Mg<sup>2+</sup>(I) (<xref ref-type="fig" rid="fig3">Figure 3B–E</xref>). The structure provides strong support to the hypothesis that GE inhibits RNAP by directly interfering with function of the i and i+1 sites and/or Mg<sup>2+</sup>(I).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02450.009</object-id><label>Figure 3.</label><caption><title>Structural basis of transcription inhibition by GE: crystal structure of RNAP-GE.</title><p>(<bold>A</bold>) Overall structure. Green, GE; violet sphere, Mg<sup>2+</sup>(I); yellow, σ. (<bold>B</bold>) Crystallographic data and refinement statistics. (<bold>C</bold>) Electron density and atomic model for GE. Blue mesh, mF<sub>o</sub>-DF<sub>c</sub> omit map for GE (contoured at 2.5σ); blue sticks, GE; gray ribbons, RNAP backbone; green surfaces, RNAP residues at which substitutions confer GE-resistance (<xref ref-type="fig" rid="fig2">Figure 2A</xref>; <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2C</xref>); cyan sticks, additional RNAP residues that contact GE; gray and red sticks additional RNAP residues that coordinate Mg<sup>2+</sup>(I); violet sphere, Mg<sup>2+</sup>(I). D2, LR, H, I, Mg<sup>2+</sup>, and BH denote the RNAP D2 loop, link region, H region, I region, Mg<sup>2+</sup> loop, and bridge helix. RNAP residues are numbered both as in <italic>T. thermophilus</italic> RNAP and as in <italic>E. coli</italic> RNAP (in parentheses). (<bold>D</bold>) Contacts between RNAP and GE (stereodiagram). Gray ribbons, RNAP backbone; gray sticks, RNAP carbon atoms; green, GE carbon atoms; red, oxygen atoms; blue, nitrogen atoms; red spheres, water molecules; violet sphere, Mg<sup>2+</sup>(I). Blue dashed lines, H-bonds; orange dashed lines, coordinate-covalent bonds. (<bold>E</bold>) Contacts between RNAP and GE (schematic). Red dashed lines, H-bonds; orange dashed lines, coordinate-covalent bonds; blue arcs, van der Waals interactions; W, water molecule. See <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.009">http://dx.doi.org/10.7554/eLife.02450.009</ext-link></p></caption><graphic xlink:href="elife02450f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.010</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Structural basis of transcription inhibition by GE.</title><p>Location of contacting residues in the sequences of RNAP β subunit (top) and RNAP β' subunit (bottom). Sequence alignments for the β and β' subunits of bacterial RNAP (top twenty-one sequences in each panel) and corresponding subunits of human RNAP I, RNAP II, and RNAP III (bottom three sequences in each panel), showing locations of residues that contact GE in the crystal structure of RNAP-GE (black rectangles; identities from <xref ref-type="fig" rid="fig3">Figure 3E</xref>), and locations of the RNAP D2 loop, link region, H region, I region, Mg<sup>2+</sup> loop, and bridge helix (black bars; boundaries from <xref ref-type="bibr" rid="bib72">Sweetser et al., 1987</xref> and <xref ref-type="bibr" rid="bib79">Weinzierl, 2010</xref>). Species names are as in <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.010">http://dx.doi.org/10.7554/eLife.02450.010</ext-link></p></caption><graphic xlink:href="elife02450fs005"/></fig></fig-group></p></sec><sec id="s2-3-2"><title>RNAP residues at which GE-resistance substitutions occur contact GE</title><p>All residues at which GE<sup>R</sup> substitutions were obtained make direct contact with GE in the crystal structure: βGlu565, βGly566, βMet681, and βAsn684 (green in <xref ref-type="fig" rid="fig3">Figure 3C</xref>). (Here and elsewhere in the text, residues are numbered as in <italic>E. coli</italic> RNAP. In <xref ref-type="fig" rid="fig3 fig4 fig5">Figures 3–5</xref>, residues are numbered both as in <italic>T. thermophilus</italic> RNAP and as in <italic>E. coli</italic> RNAP.) The sidechain of βGlu565 penetrates the GE macrocycle and makes interactions with six of seven GE residues (<xref ref-type="fig" rid="fig3">Figure 3C–E</xref>). Substitution of βGlu565 is expected to disrupt multiple H-bonds and van der Waals interactions between RNAP and GE. Substitution of βGly566 by any residue other than Gly is expected to introduce steric clash between RNAP and GE. Substitution of βMet681 is expected to disrupt van der Waals interactions between RNAP and GE. Substitution of βAsn684 is expected to disrupt H-bonds and van der Waals interactions between RNAP and GE.</p></sec><sec id="s2-3-3"><title>Additional RNAP residues contact GE</title><p>Besides the residues at which GE<sup>R</sup> substitutions were obtained, 11 additional residues—all located in the RNAP active-center i and i+1 sites—make direct interactions with GE in the crystal structure: βPro564, βAsn568, βArg678, βMet685, βGln688, βLys1065, βLys1073, βHis1237, β′Asp462, β’Thr786, and β’Ala787 (cyan in <xref ref-type="fig" rid="fig3">Figure 3C</xref>). 10 of these additional residues are invariant in RNAP from bacteria to humans (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>), and, for six, it is known that substitutions result in a loss of RNAP function (<xref ref-type="bibr" rid="bib32">Kashlev et al., 1990</xref>; <xref ref-type="bibr" rid="bib49">Mustaev et al., 1991</xref>; <xref ref-type="bibr" rid="bib56">Sagitov et al., 1993</xref>; <xref ref-type="bibr" rid="bib69">Sosunov et al., 2005</xref>; <xref ref-type="bibr" rid="bib31">Jovanovic et al., 2011</xref>). We infer that these additional residues cannot be substituted without loss of RNAP function, and thus cannot be substituted to give rise to GE-resistance.</p></sec><sec id="s2-3-4"><title>GE stereochemistry</title><p>The experimental electron density and inferred bonding patterns in the crystal structure define the stereochemistry at the five previously unassigned stereocenters of GE, as follows: D-dmaDap, D-Ser, D-Val, 3R,4S,L-dhGln, D-aThr, D-iSer, and L-Ama (<xref ref-type="fig" rid="fig3">Figure 3C–E</xref>). The assignment of stereochemistry at dhGln C3 is tentative. The assignments of stereochemistry at other stereocenters are firm.</p></sec><sec id="s2-3-5"><title>RNAP-GE interactions</title><p>The crystal structure also defines the orientation of GE relative to RNAP and the interactions between GE and RNAP (<xref ref-type="fig" rid="fig3">Figure 3C–E</xref>). GE binds within a shallow bowl-like depression formed by the D2 loop, the link region, and the Mg<sup>2+</sup> loop [Mg<sup>2+</sup>(I) and three RNAP Asp residues that coordinate Mg<sup>2+</sup>(I)], and the H and I regions (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>). GE is oriented relative to RNAP such that the GE dhGln residue is directed toward Mg<sup>2+</sup>(I) and the GE dmaDap residue is directed toward the Rif pocket (<xref ref-type="fig" rid="fig3">Figure 3C–E</xref>).</p><p>The GE dhGln residue participates in a network of interactions, including H-bonds with RNAP βGlu565, βArg678, and βLys1073, an H-bond with a water molecule in the first coordination shell of Mg<sup>2+</sup>(I), and van der Waals interactions with RNAP β′Asp462, which is one of the three RNAP Asp residues that coordinate Mg<sup>2+</sup>(I) (<xref ref-type="fig" rid="fig3">Figure 3D–E</xref>). The GE aThr residue makes an H-bond with RNAP βLys1065 and van der Waals interactions with βGlu565, βMet685, βLys1073, and βHis1237. The GE iSer residue makes H-bonds with RNAP βGlu565, βAsn684, and βGln688, and van der Waals interactions with βMet681. The GE Ama residue makes H-bonds with RNAP βGlu565 and βGln688, and van der Waals interactions with βAsn684. The GE dmaDap residue makes an H-bond with RNAP βAsn568 and van der Waals interactions with βGly566 and βPro564. Atoms of the GE dmaDap sidechain distal to the sidechain carbonyl are disordered in the structure, indicating that these atoms exhibit static or dynamic conformational heterogeneity, and suggesting that these atoms make few or no interactions with RNAP (omitted in <xref ref-type="fig" rid="fig3">Figure 3C–D</xref>; gray in <xref ref-type="fig" rid="fig3">Figure 3E</xref>). The GE Ser residue makes van der Waals interactions with RNAP βGlu565. The GE Val residue makes van der Waals interactions with RNAP βGlu565, β′Thr786, and β’Ala787.</p><p>The structure accounts for the structure-activity relationships obtained from analysis of semi-synthetic derivatives of GE. Modification of the GE dhGln sidechain eliminates RNAP-inhibitory activity (<xref ref-type="bibr" rid="bib42">Mariani et al., 2005</xref>), consistent with the participation by this sidechain in multiple H-bonds and van der Waals interactions with RNAP. Removal of the Ama sidechain reduces RNAP-inhibitory activity (<xref ref-type="bibr" rid="bib42">Mariani et al., 2005</xref>), consistent with the participation of this sidechain in an H-bond and van der Waals interactions with RNAP. Substitutions of the dmaDap sidechain acyl moiety, including substitutions with bulky groups, has little or no effect on RNAP-inhibitory activity (<xref ref-type="bibr" rid="bib42">Mariani et al., 2005</xref>), consistent with the fact that atoms of the acyl group are disordered in the structure and are inferred to make few or no interactions with RNAP.</p></sec></sec><sec id="s2-4"><title>Structural basis of inhibition by GE: crystal structure of RP<sub>o</sub>-GE</title><p>To define effects of GE on interactions of RNAP with promoter DNA, we determined a crystal structure of RP<sub>o</sub> in complex with GE at 2.8 Å resolution (<xref ref-type="fig" rid="fig4">Figure 4</xref>). The higher resolution of this structure (2.8 Å vs 3.35 Å) enables confirmation of the inferred stereochemical assignments at stereocenters of GE (<xref ref-type="fig" rid="fig4">Figure 4D,E</xref>) and enables identification of additional water-mediated H-bonds, including additional water-mediated H-bonds in the network of water-mediated interactions connecting GE to Mg<sup>2+</sup>(I) (<xref ref-type="fig" rid="fig4">Figure 4D–E</xref>; <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.02450.011</object-id><label>Figure 4.</label><caption><title>Structural basis of transcription inhibition by GE: crystal structure of RP<sub>o</sub>-GE.</title><p>(<bold>A</bold>) Overall structure. (<bold>B</bold>) Crystallographic data and refinement statistics. (<bold>C</bold>) Electron density and atomic model for GE. (<bold>D</bold>) Contacts between RP<sub>o</sub> and GE (stereodiagram). (<bold>E</bold>) Contacts between RP<sub>o</sub> and GE (schematic). See <xref ref-type="fig" rid="fig4s1 fig4s2">Figure 4—figure supplements 1, 2</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.011">http://dx.doi.org/10.7554/eLife.02450.011</ext-link></p></caption><graphic xlink:href="elife02450f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.012</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Structural basis of transcription inhibition by GE.</title><p>Network of contacts to GE dhGln residue. Stereoview. Gray, RNAP carbon atoms. Green, GE carbon atoms. Red, oxygen atoms. Blue, nitrogen atoms. Violet sphere, Mg<sup>2+</sup>(I). Red spheres, water molecules. Dashed blue lines, H-bonds. Dashed orange lines, coordinate-covalent bonds.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.012">http://dx.doi.org/10.7554/eLife.02450.012</ext-link></p></caption><graphic xlink:href="elife02450fs006"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.013</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Absence of effects of DNA on GE conformation and RNAP-GE interactions.</title><p>Superimposition of crystal structures of RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>) and RNAP-GE (<xref ref-type="fig" rid="fig3">Figure 3</xref>). Blue mesh, blue sticks, red sticks, gray surface, yellow surface, and violet sphere: mF<sub>o</sub>-DF<sub>c</sub> omit map for GE, atomic model for GE, DNA, RNAP, σ, and Mg<sup>2+</sup>(I) from crystal structure of RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Cyan sticks, atomic model for GE from crystal structure of RNAP-GE (<xref ref-type="fig" rid="fig3">Figure 3</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.013">http://dx.doi.org/10.7554/eLife.02450.013</ext-link></p></caption><graphic xlink:href="elife02450fs007"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.014</object-id><label>Figure 4—figure supplement 3.</label><caption><title>Absence of effects of GE on DNA conformation and RNAP-DNA interactions.</title><p>Superimposition of crystal structures of RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>) and RP<sub>o</sub> (<xref ref-type="bibr" rid="bib84">Zhang et al., 2012</xref>). Blue mesh, blue sticks, red sticks, gray surface, yellow surface, and violet sphere: mF<sub>o</sub>-DF<sub>c</sub> omit map for GE, atomic model for GE, DNA, RNAP, σ, and Mg<sup>2+</sup>(I) from crystal structure of RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Cyan sticks, DNA from crystal structure of RP<sub>o</sub> (<xref ref-type="bibr" rid="bib84">Zhang et al., 2012</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.014">http://dx.doi.org/10.7554/eLife.02450.014</ext-link></p></caption><graphic xlink:href="elife02450fs008"/></fig></fig-group></p><p>Comparison of the structures of RNAP-GE (<xref ref-type="fig" rid="fig3">Figure 3</xref>) and RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>) shows that promoter DNA binding to form RP<sub>o</sub> does not change the conformation of GE or the interactions between RNAP and GE (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). Comparison of structures of RP<sub>o</sub> (<xref ref-type="bibr" rid="bib84">Zhang et al., 2012</xref>) and RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>) show that GE does not change the conformation of DNA or the interactions between RNAP and DNA (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>). The results provide a graphic confirmation of the results from <xref ref-type="fig" rid="fig1">Figure 1</xref>, indicating that GE does not inhibit formation of RP<sub>o</sub> and, instead, inhibits a subsequent reaction required for the first nucleotide addition in transcription initiation.</p></sec><sec id="s2-5"><title>Relationship between GE and initiating nucleotides: mutually exclusive binding</title><sec id="s2-5-1"><title>Structural modelling of steric clash between GE and initiating nucleotides</title><p>As a first step to assess whether occupancy of the RNAP i and i+1 sites by GE interferes with the binding of nucleotides to the i and i+1 sites, we constructed a structural model of a primer-dependent transcription initiation complex by superimposing crystal structures of RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>), RP<sub>o</sub> in complex with a 2-nt RNA primer occupying the i-1 and i sites (<xref ref-type="bibr" rid="bib84">Zhang et al., 2012</xref>), and a transcription elongation complex containing an NTP in the i+1 site (<xref ref-type="bibr" rid="bib77">Vassylyev et al., 2007</xref>) (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). The resulting structural model predicts severe steric clash between GE and both the RNA 3′ nucleotide in the i site and the NTP in the i+1 site (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). The phosphate and base of the RNA 3′ nucleotide are predicted to clash with the aThr residue and Ama residue, respectively, of GE. The α-phosphate and base of the NTP in the i+1 site are predicted to clash with the dhGln residue and Val residue, respectively, of GE. The structural model strongly suggests that GE interferes with binding of nucleotides to the RNAP i and i+1 sites.</p></sec><sec id="s2-5-2"><title>Crystal structure defining interactions between RP<sub>o</sub> and initiating nucleotides in the absence of GE</title><p>As a second step to assess whether occupancy of the RNAP i and i+1 sites by GE interferes with the binding of nucleotides to the i and i+1 sites, and, in order to define how the triphosphate of the first initiating NTP interacts with RNAP and how interactions may be impacted by GE, we determined a crystal structure of RP<sub>o</sub> in complex with initiating NTPs at 3.1 Å resolution (<xref ref-type="fig" rid="fig5">Figure 5A–D</xref>). To determine the structure, we soaked a pre-formed crystal of RP<sub>o</sub> with the first initiating NTP (ATP) and a non-reactive analog of the second initiating NTP (CMPcPP). The electron density map shows unambiguous electron density for ATP in the i site and for CMPcPP:Mg<sup>2+</sup>(II) in the i+1 site (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). The resulting structure provides the first structural information of a substrate complex for de novo transcription by a multi-subunit RNAP.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.02450.015</object-id><label>Figure 5.</label><caption><title>Relationship between GE and initiating NTPs: mutually exclusive binding.</title><p>(<bold>A</bold>) Crystal structure of RP<sub>o</sub>-ATP-CMPcPP: crystallographic data and refinement statistics. (<bold>B</bold>) Crystal structure of RP<sub>o</sub>-ATP-CMPcPP: electron density and model. Green mesh, mF<sub>o</sub>-DF<sub>c</sub> omit map for ATP and CMPcPP:Mg<sup>2+</sup>(II) (contoured at 2.5σ); pink sticks, ATP and CMPcPP; red ribbon, DNA template strand; gray ribbon, RNAP bridge helix; upper and lower violet spheres, Mg<sup>2+</sup>(I) and Mg<sup>2+</sup>(II). (<bold>C</bold>) Crystal structure of RP<sub>o</sub>-ATP-CMPcPP: contacts between RNAP and initiating NTPs (stereodiagram). Gray ribbon, RNAP bridge helix; gray sticks, RNAP carbon atoms; continuous red sticks, DNA atoms; pink sticks, ATP and CMPcPP carbon atoms; individual red sticks, oxygen atoms; individual blue sticks, nitrogen atoms; red spheres, water molecules; upper and lower violet spheres, Mg<sup>2+</sup>(I) and Mg<sup>2+</sup>(II). Blue dashed lines, H-bonds; orange dashed lines, coordinate-covalent bonds. (<bold>D</bold>) Crystal structure of RP<sub>o</sub>-ATP-CMPcPP: contacts between RNAP and initiating NTPs (schematic summary). Red dashed lines, H-bonds; orange dashed lines, coordinate-covalent bonds; blue arcs, van der Waals interactions; W, water molecule; underlined residues, GE-contacting residues in RP<sub>o</sub>-GE. (<bold>E</bold>) Superimposition of crystal structures of RP<sub>o</sub>-GE and RP<sub>o</sub>-ATP-CMPcPP: inferred steric clash between GE and initiating NTPs. Blue mesh, blue sticks, red sticks, and gray ribbon: mF<sub>o</sub>-DF<sub>c</sub> omit map for GE, atomic model for GE, DNA template strand, and RNAP bridge helix from RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Pink sticks and violet spheres: ATP, CMPcPP, Mg<sup>2+</sup>(I), and Mg<sup>2+</sup>(II) from RP<sub>o</sub>-ATP-CMPcPP. (<bold>F</bold>) Crystal structure of RP<sub>o</sub>-GE plus ATP and CMPcPP: crystallographic data and refinement statistics. (<bold>G</bold>) Crystal structure of RP<sub>o</sub>-GE plus ATP and CMPcPP: electron density and model. Blue mesh, mF<sub>o</sub>-DF<sub>c</sub> omit map for GE (contoured at 2.7σ); blue sticks, GE; green mesh, mF<sub>o</sub>-DF<sub>c</sub> omit map for NTP triphosphate:Mg<sup>2+</sup>(II) in RNAP E site (contoured at 2.7σ); pink sticks, NTP triphosphate; thin pink sticks, NTP sugar and base (projected); red ribbon, DNA template strand; gray ribbon, RNAP bridge helix; upper and lower violet spheres, Mg<sup>2+</sup>(I) and Mg<sup>2+</sup>(II). See <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.015">http://dx.doi.org/10.7554/eLife.02450.015</ext-link></p></caption><graphic xlink:href="elife02450f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.016</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Relationship between GE and initiating NTPs.</title><p>Superimposition of crystal structures of RP<sub>o</sub>-GE, RP<sub>o</sub>-GpA, and the transcription elongation complex: predicted steric clash between GE and NTPs in i and i+1 sites. Blue mesh, blue sticks, red sticks, gray ribbon: mF<sub>o</sub>-DF<sub>c</sub> omit map for GE, atomic model for GE, DNA template strand, and RNAP bridge helix from crystal structure of RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Upper pink sticks and violet sphere: RNA 3' nucleotide in i site and Mg<sup>2+</sup>(I) from crystal structure of RP<sub>o</sub>-GpA (<xref ref-type="bibr" rid="bib84">Zhang et al., 2012</xref>). Lower pink sticks and violet sphere: NTP in i+1 site and Mg<sup>2+</sup>(II) from crystal structure of the transcription elongation complex (<xref ref-type="bibr" rid="bib77">Vassylyev et al., 2007</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.016">http://dx.doi.org/10.7554/eLife.02450.016</ext-link></p></caption><graphic xlink:href="elife02450fs009"/></fig></fig-group></p><p>The base and sugar moieties of the first initiating NTP make the same interactions with DNA and RNAP that the RNA 3′-nucleotide base and sugar make in a transcription elongation complex (<xref ref-type="bibr" rid="bib77">Vassylyev et al., 2007</xref>; <xref ref-type="fig" rid="fig5">Figure 5C,D</xref>). The triphosphate of the first initiating NTP extends into the space that is occupied by the RNA-1 nucleotide in a transcription elongation complex, and makes H-bonds and salt-bridges through its γ-phosphate with RNAP βGln688 and βHis1237, and through its α-phosphate with RNAP βLys1065 and βLys1073 (<xref ref-type="fig" rid="fig5">Figure 5C,D</xref>). The observed interactions of the γ-phosphate and α-phosphate with βHis1237 and βLys1065 are consistent with, and account for, crosslinking results (<xref ref-type="bibr" rid="bib49">Mustaev et al., 1991</xref>).</p><p>The base moiety of the second initiating NTP makes the same interactions with DNA and RNAP that the extending NTP base makes in an elongation complex (<xref ref-type="bibr" rid="bib77">Vassylyev et al., 2007</xref>; <xref ref-type="fig" rid="fig5">Figure 5C,D</xref>). The sugar and triphosphate of the second initiating NTP make interactions characteristic of a ‘preinsertion-mode’ elongation complex, in which the sugar and triphosphate make only a subset of the interactions required for catalysis, and, in particular, in which the triphosphate approaches, but does not coordinate, Mg<sup>2+</sup>(I) (<xref ref-type="bibr" rid="bib77">Vassylyev et al., 2007</xref>; <xref ref-type="bibr" rid="bib83">Zhang and Landick, 2009</xref>; <xref ref-type="bibr" rid="bib43">Martinez-Rucobo and Cramer, 2013</xref>; <xref ref-type="fig" rid="fig5">Figure 5C,D</xref>). The RNAP active center is not fully dehydrated and contains two ordered water molecules in the interface between the first and second initiating NTPs (red spheres in <xref ref-type="fig" rid="fig5">Figure 5C</xref>; ‘W’ in <xref ref-type="fig" rid="fig5">Figure 5D</xref>), consistent with expectation for a ‘preinsertion-mode’ complex. The RNAP trigger loop, which can adopt open or closed conformational states, adopts an open conformational state in this structure, further consistent with expectation for a ‘preinsertion-mode’ complex. It is believed that the ‘preinsertion-mode’ elongation complex is an obligatory functional intermediate in formation of the catalytically competent ‘insertion-mode’ elongation complex (<xref ref-type="bibr" rid="bib77">Vassylyev et al., 2007</xref>; <xref ref-type="bibr" rid="bib83">Zhang and Landick, 2009</xref>; <xref ref-type="bibr" rid="bib43">Martinez-Rucobo and Cramer, 2013</xref>). We suggest, by analogy, that the ‘preinsertion-mode’ initiation complex defined herein is an obligatory functional intermediate in formation of the catalytically competent ‘insertion-mode’ initiation complex.</p><p>The determination of a crystal structure of a substrate complex for de novo initiation (<xref ref-type="fig" rid="fig5">Figure 5A–D</xref>) provided a firm foundation for structural modelling of relationships between GE and initiating nucleotides in a transcription initiation complex. Accordingly, we constructed a structural model by superimposing crystal structures of RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>) and RP<sub>o</sub>-ATP-CMPcPP (<xref ref-type="fig" rid="fig5">Figure 5A–D</xref>). The resulting structural model shows severe steric clash between GE and both the first initiating NTP in the i site and the second initiating NTP in the i+1 site (<xref ref-type="fig" rid="fig5">Figure 5E</xref>). The structural model confirms the steric clashes predicted in the structural model built using an elongation complex structure (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>), and reveals new, particularly severe, steric clashes involving the triphosphate of the first initiating NTP (<xref ref-type="fig" rid="fig5">Figure 5E</xref>). The steric clashes with the triphosphate entail essentially complete steric interpenetration of the triphosphate α, β, and γ phosphates with the GE aThr and Ama residues. The structural model very strongly suggests that GE interferes with binding of nucleotides to the RNAP i and i+1 sites.</p></sec><sec id="s2-5-3"><title>Crystal structure defining interactions between RP<sub>o</sub> and initiating nucleotides in the presence of GE</title><p>To test directly whether GE interferes with binding of initiating NTPs to the i and i+1 sites, we compared NTP occupancies of the i and i+1 sites in the absence of GE to those in the presence of GE. To do this, we compared electron density maps for crystals of RP<sub>o</sub> soaked with ATP and CMPcPP (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>) to electron density maps for crystals of RP<sub>o</sub> first soaked with GE and then soaked with ATP and CMPcPP (<xref ref-type="fig" rid="fig5">Figure 5F,G</xref>). As described above, electron density maps obtained by soaking a crystal of RP<sub>o</sub> with ATP and CMPcPP show unambiguous electron density for ATP and CMPcPP in the i and i+1 sites (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). In contrast, electron density maps obtained by soaking a crystal of RP<sub>o</sub> first with GE, and then with ATP and CMPcPP show unambiguous electron density for GE, but show no density for ATP or CMPcPP in the i and i+1 sites (<xref ref-type="fig" rid="fig5">Figure 5G</xref>). Instead, electron density attributable to an NTP triphosphate is seen in a region adjacent to the i+1 site termed the ‘E site’ (<xref ref-type="fig" rid="fig5">Figure 5G</xref>). The E site previously has been reported as a binding site for a non-complementary NTP and has been proposed to serve as an entry site for NTPs on the pathway of NTP binding (<xref ref-type="bibr" rid="bib81">Westover et al., 2004</xref>). The pair of structures indicating that initiating NTPs occupy the i and i+1 sites in the absence of GE (<xref ref-type="fig" rid="fig5">Figure 5B</xref>), but do not occupy the i and i+1 sites in the presence of GE (<xref ref-type="fig" rid="fig5">Figure 5G</xref>), show graphically that GE interferes with binding of initiating NTPs to the i and i+1 sites.</p><p>In further work, we performed analogous crystal-soaking experiments to assess effects of GE on occupancy of 3′-deoxy-3′-amino-ATP and CTP (a non-reactive analog of the first initiating NTP and a reactive second initiating NTP) and of ATP and CTP (a reactive first initiating NTP and a reactive second initiating NTP). In these cases, soaking of nucleotides into RP<sub>o</sub> in the absence of GE yielded, respectively, an ‘insertion mode’ substrate complex with nucleotides in the i and i+1 sites, and a product complex with a 2-nt RNA product (to be published elsewhere). In contrast, in each case, soaking nucleotides into RP<sub>o</sub> pre-soaked with GE yielded a complex with electron density for GE, no electron density for nucleotides in the i and i+1 sites, and density attributable to an NTP triphosphate in the E site.</p><p>We conclude that GE interferes with binding of initiating nucleotides to the RNAP i and i+1 sites.</p></sec></sec><sec id="s2-6"><title>Relationship between GE and Rif: simultaneous binding</title><sec id="s2-6-1"><title>Partial-competitive binding of GE and Rif</title><p>The observation that the GE target is adjacent to the Rif target (<xref ref-type="fig" rid="fig2">Figure 2D</xref>) raises the possibility that binding of GE to RNAP may affect binding of Rif to RNAP. As a first step to assess interactions between GE and Rif, we performed fluorescence-detected binding experiments (<xref ref-type="bibr" rid="bib20">Feklistov et al., 2008</xref>) monitoring RNAP-Rif interaction in the absence and presence of GE.</p><p>The results in <xref ref-type="fig" rid="fig6">Figure 6A–C</xref> show that GE inhibits the binding of Rif to RNAP. GE decreases k<sub>on</sub> for Rif ∼20-fold, increases k<sub>off</sub> for Rif ∼fourfold, and increases the equilibrium dissociation constant (K<sub>d</sub>) for Rif ∼80-fold (<xref ref-type="fig" rid="fig6">Figure 6C</xref>). The equilibrium dissociation constant for inhibition of RNAP-Rif interaction by GE (K<sub>i</sub>) is 6 nM, which is comparable to the IC50 for inhibition of RNAP by GE (<xref ref-type="fig" rid="fig6">Figure 6A</xref>; <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>; <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1B</xref>). GE<sup>R</sup> RNAP derivatives do not exhibit inhibition of RNAP-Rif interaction by GE, indicating that the inhibition requires specific interactions of GE with the GE target (<xref ref-type="fig" rid="fig6">Figure 6B</xref>).<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.02450.017</object-id><label>Figure 6.</label><caption><title>Relationship between GE and Rif: simultaneous binding.</title><p>(<bold>A</bold>) Partial-competitive binding of GE and Rif: association kinetics for Rif in presence of 0–2000 nM GE. (<bold>B</bold>) Partial-competitive binding of GE and Rif: association kinetics for Rif in presence of 2000 nM GE, using wild-type RNAP (red) and GE-resistant RNAP derivatives [Asp565]β-RNAP (black) and [Lys684]β-RNAP (blue). (<bold>C</bold>) Partial-competitive binding of GE and Rif: k<sub>on</sub>, k<sub>off</sub>, and K<sub>d</sub> for Rif in absence of GE and in presence of saturating GE (160 nM or 2000 nM; ∼30 × K<sub>i</sub> or ∼300 × K<sub>i</sub>). (<bold>D</bold>) Superimposition of crystal structures of RP<sub>o</sub>-GE and RNAP-Rif: inferred simultaneous binding. Blue mesh, blue sticks, gray ribbon, and violet sphere: mF<sub>o</sub>-DF<sub>c</sub> omit map for GE, atomic model for GE, RNAP, and Mg<sup>2+</sup>(I) from RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Yellow sticks: Rif from RNAP-Rif (PDB: 1YNN). Green surfaces, GE target residues at which substitutions confer GE-resistance; red surfaces, residues at which substitutions confer Rif-resistance. (<bold>E</bold>) Crystal structure of RP<sub>o</sub>-GE plus Rif: crystallographic data and refinement statistics. (<bold>F</bold>) Crystal structure of RP<sub>o</sub>-GE plus Rif: electron density and model. Yellow mesh, patchy electron density potentially attributable to Rif (mF<sub>o</sub>-DF<sub>c</sub> omit map; contoured at 2.7σ). Other colors as in <bold>D</bold>. (<bold>G</bold>) Crystal structure of RP<sub>o</sub>-GE-RifSV: crystallographic data and refinement statistics. (<bold>H</bold>) Crystal structure of RP<sub>o</sub>-GE-RifSV: electron density and model. Yellow mesh, mF<sub>o</sub>-F<sub>c</sub> omit map for RifSV (contoured at 2.7σ); yellow sticks, RifSV. Other colors as in <bold>D</bold>. See <xref ref-type="fig" rid="fig6s1 fig6s2">Figure 6—figure supplements 1, 2</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.017">http://dx.doi.org/10.7554/eLife.02450.017</ext-link></p></caption><graphic xlink:href="elife02450f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.018</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Relationship between GE and Rif: effects of GE on RNAP-Rif interaction.</title><p>Left panel, association kinetics: I<sub>∞</sub> for RNAP-Rif interaction in the presence of 0, 2.5, 10, 40, 160, or 2000 nM GE. Center panel, association kinetics: k<sub>obs</sub> for RNAP-Rif interaction in the presence of 0 or 2000 nM GE. Right panel, dissociation kinetics: I for RNAP-Rif interaction in the presence of 0 or 2000 nM GE.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.018">http://dx.doi.org/10.7554/eLife.02450.018</ext-link></p></caption><graphic xlink:href="elife02450fs010"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02450.019</object-id><label>Figure 6—figure supplement 2.</label><caption><title>Relationship between GE and Rif: superimposition of crystal structures of RP<sub>o</sub>-GE and RP<sub>o</sub>-GE-RifSV showing differences in conformations of GE dmaDap residue.</title><p>Blue sticks, yellow sticks, and blue numbers: GE, RifSV, and distances between GE dmaDap sidechain carbonyl carbon and RifSV C3 and O<sup>4</sup> atoms, in RP<sub>o</sub>-GE-RifSV. Gray sticks and gray numbers: GE in RP<sub>o</sub>-GE and corresponding distances calculated for the GE conformation in RP<sub>o</sub>-GE.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.019">http://dx.doi.org/10.7554/eLife.02450.019</ext-link></p></caption><graphic xlink:href="elife02450fs011"/></fig></fig-group></p><p>However, the results in <xref ref-type="fig" rid="fig6">Figure 6A–C</xref> also show that GE does not preclude the binding of Rif to RNAP. Thus, even at saturating concentrations of GE, RNAP-Rif interaction still occurs (<xref ref-type="fig" rid="fig6">Figure 6A</xref>) and still exhibits a submicromolar K<sub>d</sub> (K<sub>d</sub> = 30 nM; <xref ref-type="fig" rid="fig6">Figure 6C</xref>).</p><p>The results quantitatively fit a model of partial competitive binding—i.e., a model in which X inhibits the binding of Y, but in which X and Y can bind simultaneously at sufficient concentrations; <xref ref-type="bibr" rid="bib65">Segel, 1975</xref>. We infer that GE inhibits the binding of Rif to RNAP, but that GE and Rif can bind simultaneously to RNAP at sufficient concentrations. The observation that GE inhibits the binding of Rif is consistent with the fact that the GE target is adjacent to the Rif target (<xref ref-type="fig" rid="fig2">Figure 2D</xref>), enabling steric clash between GE bound to the GE target and Rif bound to the Rif target. The observation that GE does not preclude the binding of Rif is consistent with the observation that the GE target does not overlap with the Rif target (<xref ref-type="fig" rid="fig2">Figure 2D–F</xref>).</p></sec><sec id="s2-6-2"><title>Structural modelling of simultaneous binding of GE and a rifamycin</title><p>As a next step to assess interactions between GE and Rif, we constructed a structural model of GE bound to the GE target and Rif bound to the Rif target. To construct the model, we superimposed the crystal structure of RP<sub>o</sub>-GE (<xref ref-type="fig" rid="fig4">Figure 4</xref>) on a crystal structure of RNAP-Rif (<xref ref-type="bibr" rid="bib7">Campbell et al., 2005</xref>). The structural model predicts that GE bound to the GE target is located immediately adjacent to Rif bound to the Rif target (<xref ref-type="fig" rid="fig6">Figure 6D</xref>). The structural model further predicts that there is steric clash between GE bound to the GE target and Rif bound to the Rif target, but that clash is limited to the dmaDap sidechain of GE and the C3 atom and sidechain of Rif (cyan in <xref ref-type="fig" rid="fig6">Figure 6D</xref>). The predicted adjacent binding and steric clash are consistent with the observation that GE and Rif compete for binding (<xref ref-type="fig" rid="fig6">Figure 6A–C</xref>). The predicted limitation of the steric clash to a single moiety of GE and a single moiety of Rif is consistent with the observation that GE and Rif can bind simultaneously to RNAP at sufficient concentrations (<xref ref-type="fig" rid="fig6">Figure 6A–C</xref>).</p></sec><sec id="s2-6-3"><title>Crystal structures defining simultaneous binding of GE and a rifamycin</title><p>As a next step to assess interactions between GE and rifamycins, we sought to determine crystal structures of RP<sub>o</sub> bound simultaneously to GE and a rifamycin. In a first effort, we soaked crystals of RP<sub>o</sub> with GE and Rif (<xref ref-type="fig" rid="fig6">Figure 6E,F</xref>). The resulting electron density maps showed unambiguous electron density for GE in the GE target, but only limited density in the Rif target (<xref ref-type="fig" rid="fig6">Figure 6F</xref>). In a second effort, noting that steric clash may be limited to the dmaDap sidechain of GE and the C3 atom and sidechain of Rif (<xref ref-type="fig" rid="fig6">Figure 6D</xref>), we soaked crystals of RP<sub>o</sub> with GE and rifamycin SV (RifSV), a Rif analog that lacks the C3 sidechain and that retains high RNAP-inhibitory and antibacterial potency (<xref ref-type="fig" rid="fig6">Figure 6G–H</xref>; <xref ref-type="bibr" rid="bib66">Sensi et al., 1966</xref>). In this case, the resulting electron density maps showed unambiguous electron density for GE in the GE target and for RifSV in the Rif target (<xref ref-type="fig" rid="fig6">Figure 6H</xref>). Occupancy levels for both GE and RifSV were 1, indicating that GE and RifSV were bound simultaneously to RNAP in the crystal. The inability to obtain a structure with simultaneously bound ligands upon crystal soaking with GE and Rif, but ability to obtain a structure with simultaneously bound ligands upon crystal soaking with GE and RifSV, highlights the contribution of the rifamycin C3 region to steric clash between GE and rifamycins.</p><p>The conformation of the GE dmaDap residue differs in RP<sub>o</sub>-GE and RP<sub>o</sub>-GE-RifSV (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>). The GE dmaDap sidechain in RP<sub>o</sub>-GE-RifSV is rotated by ∼110°, in a direction that increases the distance between the dmaDap sidechain carbonyl carbon and the RifSV C3 atom from 3.7 Å to 8.6 Å and thereby alleviates steric clash. This observation highlights the contribution of the GE dmaDap residue to steric clash between GE and rifamycins.</p></sec></sec><sec id="s2-7"><title>Bipartite inhibitors: GE-rifamycin and GE-sorangicin</title><sec id="s2-7-1"><title>Structural modelling of GE-rifamycin and GE-sorangicin bipartite inhibitors</title><p>The crystal structure of RP<sub>o</sub>-GE-RifSV immediately suggests the possibility of constructing a bipartite compound comprising GE, linked through its dmaDap residue, to a rifamycin, linked through its C3 or O<sup>4</sup> atom (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). Fortuitously, the GE dmaDap residue is one of three GE residues that have chemical reactivity that can be, and has been, exploited for derivatization by semi-synthesis (sole α,β-unsaturated amide moiety in GE; enables site-selective hydrolysis, ozonolysis, and 1,4-addition; <xref ref-type="bibr" rid="bib42">Mariani et al., 2005</xref>; YWE and RHE, unpublished), and the rifamycin C3 and O<sup>4</sup> atoms have chemical reactivities that can be, and extensively have been, exploited for derivatization of rifamycins by semi-synthesis (<xref ref-type="bibr" rid="bib66">Sensi et al., 1966</xref>). Still more fortuitously, the GE dmaDap residue and the rifamycin C3 and O<sup>4</sup> atoms are positions that can be modified without loss of activity (<xref ref-type="bibr" rid="bib66">Sensi et al., 1966</xref>; <xref ref-type="bibr" rid="bib42">Mariani et al., 2005</xref>). Accordingly, synthesis of such a bipartite compound not only is possible, but also is tractable. Such a bipartite compound is expected to be able to bind simultaneously to the GE target (through the GE moiety) and the Rif target (through the rifamycin moiety). Accordingly, such a compound is expected to have exceptionally high binding affinity, exceptionally high RNAP-inhibitory potency, and an ability to overcome resistance arising from substitutions in one of the GE target and the Rif target.<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.02450.020</object-id><label>Figure 7.</label><caption><title>Bipartite inhibitors: GE-rifamycin and GE-sorangicin.</title><p>(<bold>A</bold>) Proposed bipartite inhibitor having GE dmaDap sidechain linked to RifSV C3 or O<sup>4</sup> atom. Crystal structure of RP<sub>o</sub>-GE-RifSV. Black line, linker. Other colors as in <xref ref-type="fig" rid="fig6">Figure 6H</xref>. (<bold>B</bold>) Proposed bipartite inhibitor having GE dmaDap sidechain linked to Sor sidechain carboxyl. Superimposition of crystal structures of RPo-GE and RNAP-Sor (PDB: 1YNJ). Cyan, Sor; red, residues at which substitutions confer Sor-resistance. Other colors as in <bold>A</bold>. (<bold>C</bold>) Synthesis of bipartite inhibitor having GE dmaDap sidechain linked to RifSV C3 atom (RifaGE-3). (<bold>D</bold>) Inhibition of GE-resistant RNAP ([Asp565]β-RNAP) by RifaGE-3. (<bold>E</bold>) Inhibition of Rif-resistant RNAP ([Asn516]β-RNAP) by RifaGE-3.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.020">http://dx.doi.org/10.7554/eLife.02450.020</ext-link></p></caption><graphic xlink:href="elife02450f007"/></fig></p><p>Sor, a compound not structurally-related to rifamycins, functions by binding to the Rif binding site (<xref ref-type="bibr" rid="bib7">Campbell et al., 2005</xref>; <xref ref-type="bibr" rid="bib27">Ho et al., 2009</xref>). Structural modelling of RP<sub>o</sub> having GE bound to the GE target and Sor bound to the Rif binding site (<xref ref-type="fig" rid="fig7">Figure 7B</xref>), indicates that GE and Sor, like GE and a rifamycin, may be able to bind simultaneously to RNAP, and may be able to be linked to yield a bipartite inhibitor with exceptionally high binding affinity, exceptionally high RNAP-inhibitory potency, and an ability to overcome resistance arising from substitutions in one of the GE target and the Sor target. Fortuitously, the part of Sor that is predicted to be closest to, and potentially linkable, to GE is the Sor carboxyl moiety (<xref ref-type="fig" rid="fig7">Figure 7B</xref>), which has chemical reactivity that can be, and has been, exploited for derivatization of Sor by semi-synthesis, and which can be modified without loss of RNAP-inhibitory activity and antibacterial activity (<xref ref-type="bibr" rid="bib29">Jansen et al., 1990</xref>).</p></sec><sec id="s2-7-2"><title>Synthesis and evaluation of a GE-rifamycin bipartite inhibitor</title><p>We have synthesized and evaluated a bipartite inhibitor comprising a GE derivative and RifSV, covalently connected through the GE-derivative dmaDap sidechain, the RifSV C3 atom, and a one-atom linker (‘RifaGE-3’; compound 3 of <xref ref-type="fig" rid="fig7">Figure 7C</xref>). To prepare the bipartite inhibitor, we employed a three-step procedure involving: (a) site-selective introduction of an amino group into the GE dmaDap sidechain through 1,4-addition (with concomitant heat/acid-catalyzed decarboxylation of the GE Ama sidechain), (b) reaction with 3-bromo-rifamycin S, and (c) reduction with sodium ascorbate (<xref ref-type="fig" rid="fig7">Figure 7C</xref>). The resulting bipartite inhibitor inhibits wild-type RNAP with an IC50 at the limit of detection of the assay (IC50 ≤40 nM), inhibits GE<sup>R</sup> RNAP &gt;2500-fold more potently than GE (<xref ref-type="fig" rid="fig7">Figure 7D</xref>), and inhibits Rif<sup>R</sup> RNAP 50-fold more potently than RifSV (<xref ref-type="fig" rid="fig7">Figure 7E</xref>). The biochemical, microbiological, and structural characterization of the bipartite inhibitor, as well as the optimization of linkage sites, linker lengths, and synthetic methods for preparation of bipartite inhibitors, will be reported separately. Nevertheless, the results in <xref ref-type="fig" rid="fig7">Figure 7C–E</xref> provide proof-of-concept for the synthesis, the high potency against wild-type RNAP, and the ability to overcome resistance of a GE-rifamycin bipartite inhibitor.</p></sec></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Our results establish GE inhibits RNAP through a novel mechanism and a novel target. Our results show that GE inhibits the first nucleotide-addition step in transcription initiation (<xref ref-type="fig" rid="fig1">Figure 1</xref>), show that GE functions through a binding site that overlaps the RNAP active-center i and i+1 sites (<xref ref-type="fig" rid="fig2">Figure 2</xref>), define the structural basis of RNAP-GE interaction and RP<sub>o</sub>-GE interaction (<xref ref-type="fig" rid="fig3 fig4">Figures 3,4</xref>), and show that GE prevents binding of initiating NTPs to the RNAP i and i+1 sites (<xref ref-type="fig" rid="fig5">Figure 5</xref>).</p><p>Our results further establish that the binding site on RNAP for GE is adjacent to, but does not substantially overlap, the binding site on RNAP for the rifamycin antibacterial drugs (<xref ref-type="fig" rid="fig2">Figure 2D–F</xref>), show that GE and a rifamycin can bind simultaneously to their adjacent binding sites in RNAP (<xref ref-type="fig" rid="fig6">Figure 6</xref>), and show that GE and a rifamycin can be covalently linked, through the GE dmaDap sidechain and the rifamycin C3-O<sup>4</sup> region, to yield a bipartite RNAP inhibitor that binds to both the GE target and the rifamycin target (<xref ref-type="fig" rid="fig7">Figure 7</xref>).</p><p>Three features of the GE target, identified in this work, indicate that the GE target is an unusually attractive target—a ‘privileged target’—for antibacterial drug discovery involving RNAP. First, since most residues of the GE binding site are functionally critical residues of the RNAP active center that cannot be substituted without loss of RNAP activity, the target-based resistance spectra of an antibacterial compound that functions through the GE binding site will be small (∼1/10 the size of the target-based resistance spectrum of Rif; ∼1/10 to ∼1/5 the size of the target-based resistance spectra of RNAP inhibitors; <xref ref-type="fig" rid="fig2">Figure 2D</xref>; <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). Second, since the GE binding site is different from the rifamycin binding site, an antibacterial compound that functions through the GE binding site will not exhibit target-based cross-resistance with rifamycins (<xref ref-type="fig" rid="fig2">Figure 2E,F</xref>; <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2D,E</xref>). Third, since the GE binding site is adjacent to, but does not substantially overlap, the rifamycin binding site (<xref ref-type="fig" rid="fig2 fig6">Figures 2D and 6</xref>), an antibacterial compound that functions through the GE binding site can be linked to a rifamycin or a sorangicin to construct a bipartite, bivalent inhibitor that binds to both the GE target and the rifamycin target and, therefore, that is exceptionally potent and exceptionally refractory to target-based resistance (<xref ref-type="fig" rid="fig7">Figure 7</xref>).</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>RNAP inhibitors</title><p>GE23077 (GE) was prepared from cultures of <italic>Actinomadura</italic> sp. DSMZ 13491 as in <xref ref-type="bibr" rid="bib10">Ciciliato et al. (2004)</xref>. Sorangicin (Sor) was prepared from cultures of <italic>Sorangium cellulosum</italic> strain So cel2 as in <xref ref-type="bibr" rid="bib28">Irschik et al. (1987)</xref>. Lipiarmycin (Lpm) was prepared from cultures of <italic>Actinoplanes deccanensis</italic> as in <xref ref-type="bibr" rid="bib12">Coronelli et al. (1975)</xref>. (±)E,E-myxopyronin B (Myx) was synthesized as in <xref ref-type="bibr" rid="bib16">Ebright and Ebright (2013)</xref>. Rifampin (Rif), rifamycin SV (RifSV), streptolydigin (Stl), and CBR703 were purchased from Sigma–Aldrich (St. Louis, MO), Sigma–Aldrich, Sourcon-Padena (Tübingen, Germany), and Maybridge (Tintagel, UK), respectively.</p></sec><sec id="s4-2"><title>Plasmids</title><p>Plasmid pRL706 encodes C-terminally hexahistidine-tagged <italic>E. coli</italic> RNAP β subunit under control of the <italic>trc</italic> promoter (<xref ref-type="bibr" rid="bib62">Severinov et al., 1997</xref>). Plasmid pRL663 encodes C-terminally hexahistidine-tagged <italic>E. coli</italic> RNAP β′ subunit under control of the <italic>tac</italic> promoter (<xref ref-type="bibr" rid="bib78">Wang et al., 1995</xref>). Plasmid pKD46 carries a temperature-sensitive replication origin, confers ampicillin-resistance, and encodes λ Exo, Beta, and Gam, under control of the <italic>P</italic><sub><italic>araB</italic></sub> promoter (<xref ref-type="bibr" rid="bib13">Datsenko and Wanner, 2000</xref>). Plasmid pAKE604 confers kanamycin-resistance and sucrose-sensitivity (<xref ref-type="bibr" rid="bib17">El-Sayed et al., 2001</xref>).</p></sec><sec id="s4-3"><title><italic>E. coli</italic> RNAP holoenzyme</title><p><italic>E. coli</italic> RNAP, [Asn516]β-RNAP, [Asp565]β-RNAP, and [Lys684]β-RNAP core and holoenzyme were prepared from <italic>E. coli</italic> strain XE54 (<xref ref-type="bibr" rid="bib73">Tang et al., 1994</xref>) transformed with pRL706, pRL706-516N, pRL706-565D, and pRL706-684K, respectively, using procedures essentially as in <xref ref-type="bibr" rid="bib51">Niu et al. (1996)</xref>. <italic>E. coli</italic> RNAP holoenzyme derivatives site-specifically labelled with fluorescein at σ<sup>70</sup> residue 517 ([F<sup>517</sup>]σ<sup>70</sup>-RNAP holoenzyme derivatives) were prepared as in <xref ref-type="bibr" rid="bib33">Knight et al. (2005)</xref>.</p></sec><sec id="s4-4"><title><italic>T. thermophilus</italic> RNAP holoenzyme</title><p><italic>T. thermophilus</italic> RNAP holoenzyme was prepared as in <xref ref-type="bibr" rid="bib84">Zhang et al. (2012)</xref>.</p></sec><sec id="s4-5"><title>RNAP-inhibitory activity</title><p>Fluorescence-detected RNAP-inhibition assays were performed by a modification of the procedure of <xref ref-type="bibr" rid="bib34">Kuhlman et al. (2004)</xref>. Reaction mixtures contained (20 μl): 0–100 μM test compound, bacterial RNAP holoenzyme (75 nM <italic>E. coli</italic> RNAP holoenzyme or <italic>E. coli</italic> RNAP holoenzyme derivative, 75 nM <italic>Staphylococcus aureus</italic> RNAP core enzyme and 300 nM <italic>S. aureus</italic> σ<sup>A</sup> [prepared as in <xref ref-type="bibr" rid="bib70">Srivastava et al. 2011</xref>], 75 nM <italic>Mycobacterium tuberculosis</italic> RNAP core enzyme and 300 nM <italic>M. tuberculosis</italic> σ<sup>A</sup> [prepared as in <xref ref-type="bibr" rid="bib70">Srivastava et al. 2011</xref>], or 75 nM <italic>T. thermophilus</italic> RNAP holoenzyme), 20 nM DNA fragment containing the bacteriophage T4 N25 promoter (positions −72 to +367; prepared by PCR from plasmid pARTaqN25-340-tR2 [<xref ref-type="bibr" rid="bib39">Liu, 2007</xref>]), 100 μM ATP, 100 μM GTP, 100 μM UTP, and 100 μM CTP, in TB (50 mM Tris–HCl, pH 8.0, 100 mM KCl, 10 mM MgCl<sub>2</sub>, 1 mM DTT, 10 μg/ml bovine serum albumin, 5% methanol, and 5.5% glycerol). Reaction components other than DNA and NTPs were pre-incubated 10 min at 37°C. Reactions were carried out by addition of DNA and incubation 15 min at 37°C, followed by addition of NTPs and incubation 60 min at 37°C. DNA was removed by addition of 1 μl 5 mM CaCl<sub>2</sub> and 2 U DNase I (Ambion, Grand Island, NY), followed by incubation 90 min at 37°C. RNA was quantified by addition of 100 μl Quant-iT RiboGreen RNA Reagent (Life Technologies, Grand Island, NY; 1:500 dilution in 10 mM Tris–HCl, pH 8.0, 1 mM EDTA), followed by incubation 10 min at 22°C, followed by measurement of fluorescence intensity (excitation wavelength = 485 nm and emission wavelength = 535 nm; GENios Pro microplate reader [Tecan, Männedorf, Switzerland]).</p><p>Radiochemical assays with human RNAP I/II/III were performed essentially as in <xref ref-type="bibr" rid="bib59">Sawadogo and Roeder (1985)</xref>. Reaction mixtures contained (20 µl): 0–100 µM GE, 8 U HeLaScribe Nuclear Extract (Promega, Madison, WI), 1 µg human placental DNA (Sigma–Aldrich), 400 μM ATP, 400 μM [α<sup>32</sup>P]UTP (0.11 Bq/fmol), 400 μM CTP, 400 μM GTP, 50 mM Tris–HCl, pH 8.0, 7 mM HEPES-NaOH, 70 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 50 mM KCl, 12 mM MgCl<sub>2</sub>, 5 mM DTT, 0.1 mM EDTA, 0.08 mM phenylmethylsulfonyl fluoride, and 16% glycerol. Reaction components other than DNA and NTPs were pre-incubated 10 min at 30°C, DNA was added and reaction mixtures were incubated 15 min at 30°C, NTPs were added and reaction mixtures were incubated 60 min at 30°C. Reaction mixtures were spotted on DE81 filter discs (Whatman, Kent, UK; pre-wetted with water) and incubated 1 min at room temperature. Filters were washed with 3 × 3 ml Na<sub>2</sub>HPO<sub>4</sub>, 2 × 3 ml water, and 3 ml ethanol, using a filter manifold (Hoefer, Holliston, MA). Filters were placed in scintillation vials containing 10 ml Scintiverse BD Cocktail (Thermo Fisher, Waltham, MA), and radioactivity was quantified by scintillation counting (LS6500; Beckman–Coulter, Brea, CA).</p><p>Half-maximal inhibitory concentrations (IC50s) were calculated by non-linear regression in SigmaPlot (SPSS, Chicago, IL).</p></sec><sec id="s4-6"><title>Antibacterial activity</title><p>Minimum inhibitory concentrations (MICs) were quantified using broth microdilution assays (<xref ref-type="bibr" rid="bib11">Clinical and Laboratory Standards Institute, 2009</xref>), using a starting cell density of 3 × 10<sup>4</sup> cfu/ml, LB broth (<xref ref-type="bibr" rid="bib57">Sambrook and Russell, 2001</xref>), and an air atmosphere for <italic>E. coli</italic> D21f2tolC (<italic>tolC:</italic>Tn<italic>10 rfa lac28 proA23 trp30 his51 rpsL173 ampC tsx81</italic>; strain with cell-envelope defects resulting in increased susceptibility to hydrophobic agents, including GE; Fralick and Burns-Keliher, 1994; unpublished data), and using a starting cell density of 3 × 10<sup>4</sup> cfu/ml, Bacto Todd Hewitt broth (TH broth; BD Biosciences, San Jose, CA), and a 7% CO<sub>2</sub>/6% O<sub>2</sub>/4% H<sub>2</sub>/83% N<sub>2</sub> atmosphere for <italic>S. pyogenes</italic> and <italic>M. catarrhalis</italic>.</p></sec><sec id="s4-7"><title>GE-resistant mutants: isolation and sequencing</title><p>Saturation mutagenesis of <italic>rpoB</italic> plasmid pRL706 and <italic>rpoC</italic> plasmid pRL663 was performed by use of PCR amplification with ‘doped’ oligodeoxyribonucleotide primers (methods as in <xref ref-type="bibr" rid="bib46">Mukhopadhyay et al. 2008</xref>). ‘Doped’ oligodeoxyribonucleotide primers corresponding to codons 136-143, 504-511, 512-522, 523-534, 535-541, 542-549, 563-573, 677-690, 758-763, 813-814, 829-835, 1054-1060, 1064-1074, 1102-1108, and 1233-1242 of the <italic>rpoB</italic> gene of plasmid pRL706, and codons 347-355, 425-429, 456-465, 779-792, and 934-943 of the <italic>rpoC</italic> gene of plasmid pRL663, were synthesized on an Applied Biosystems 392/394 automated DNA/RNA synthesizer (Foster City, CA) using solid-phase β-cyanoethylphosphoramidite chemistry (sequences in <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2A</xref>). The level of ‘doping’ (nucleotide misincorporation) was selected to yield an average of 0.4–1 substitution(s) per molecule of oligodeoxyribonucleotide primer (equations in <xref ref-type="bibr" rid="bib26">Hermes et al., 1989</xref>, <xref ref-type="bibr" rid="bib25">1990</xref>). Thus, the nucleotides corresponding to codons 758-763 and 813-814 of <italic>rpoB</italic>, and codons 425-429 of <italic>rpoC</italic> were synthesized using phosphoramidite reservoirs containing 92% of the correct phosphoramidite and 8% of a 1:1:1:1 mix of dA, dC, dG, and dT phosphoramidites (i.e., 94% total correct phosphoramidite and 6% total incorrect phosphoramidite). The nucleotides corresponding to codons 136-143, 504-511, 512-522, 523-534, 535-541, 542-549, 563-573, 677-690, 829-835, 1054-1060, 1064-1074, 1102-1108, and 1233-1242 of <italic>rpoB</italic>, and codons 347-355, 456-465, 779-792, and 934-943 of <italic>rpoC</italic> were synthesized using phosphoramidite reservoirs containing 98% of the correct phosphoramidite and 2% of a 1:1:1:1 mix of dA, dC, dG, and dT phosphoramidites, (i.e., 98.5% total correct phosphoramidite and 1.5% total incorrect phosphoramidite.) All other nucleotides were synthesized using phosphoramidite reservoirs containing 100% of the correct phosphoramidite. Mutagenesis reactions were performed using the QuikChange XL Site-Directed Mutagenesis Kit (Agilent/Stratagene, La Jolla, CA) with a “doped” oligodeoxyribonucleotide primer, a complementary oligodeoxyribonucleotide primer, and pRL706 or pRL663 as template (primers at 75-150 nM; all other components at concentrations as specified by the manufacturer). Mutagenized plasmid DNA was introduced by transformation into <italic>E. coli</italic> XL1-Blue (Agilent/Stratagene). Transformants (10<sup>3</sup>-10<sup>4</sup> cells) were applied to LB-agar plates (<xref ref-type="bibr" rid="bib57">Sambrook and Russell, 2001</xref>) containing 200 μg/ml ampicillin, plates were incubated 16 hr at 37°C, and plasmid DNA was prepared from the pooled resulting colonies. The resulting passaged mutagenized plasmid libraries for the 15 “doped” oligonucleotide primers targeting <italic>rpoB</italic> were pooled on an equimolar basis, and the resulting passaged mutagenized plasmid libraries for the five “doped” oligonucleotide primers targeting <italic>rpoC</italic> were pooled on an equimolar basis. Pooled, passaged mutagenized plasmid libraries for each gene were introduced by transformation into <italic>E. coli</italic> D21f2tolC. Transformants (∼10<sup>3</sup> cells) were applied to LB-agar plates containing 200-500 μg/ml GE, 200 μg/ml ampicillin, and 1 mM IPTG; and plates were incubated 24-48 hr at 37°C. GE-resistant mutants were identified by the ability to form colonies on this medium, were confirmed by re-streaking on the same medium, were further confirmed by quantifying resistance levels in liquid cultures and accepting only isolates with &gt;2-fold resistance (procedures as described below), and were demonstrated to contain plasmid-linked GE-resistant mutations by preparing plasmid DNA, transforming <italic>E. coli</italic> D21f2tolC with plasmid DNA, and plating transformants on the same medium. For each confirmed mutant, nucleotide sequences of <italic>rpoB</italic> and <italic>rpoC</italic> were determined by Sanger sequencing (eight primers per gene).</p></sec><sec id="s4-8"><title>GE-resistant mutants: complementation assays</title><p>Temperature-sensitive <italic>E. coli</italic> strain RL585 [<italic>rpoB</italic><sup><italic>am</italic></sup><italic>cI supD</italic><sup><italic>ts</italic></sup><italic>43,74</italic> Δ<italic>(recA-srl)306 lacZ</italic><sup><italic>am</italic></sup><italic>2110 galEK</italic><sup><italic>am</italic></sup> <italic>leu</italic><sup><italic>am</italic></sup> <italic>trp</italic><sup><italic>am</italic></sup> <italic>sueA rpsL tsx srl301</italic>::Tn<italic>10-84</italic>; <xref ref-type="bibr" rid="bib35">Landick et al., 1990</xref>] was transformed with pRL706 or a pRL706 derivative, transformants (10<sup>3</sup>-10<sup>4</sup> cells) were applied to LB-agar plates containing 200 μg/ml ampicillin, 1 mM IPTG, and 10 μg/ml tetracycline, plates were incubated 22 hr at 43°C, and bacterial growth was scored.</p></sec><sec id="s4-9"><title>GE-resistant mutants: transfer to chromosome</title><p>GE-resistant and Rif-resistant mutations were transferred from pRL706 derivatives to the chromosome of <italic>E. coli</italic> D21f2tolC by λ-Red-mediated recombineering (procedures analogous to those in <xref ref-type="bibr" rid="bib13">Datsenko and Wanner 2000</xref> and <xref ref-type="bibr" rid="bib60">Sawitzke et al., 2007</xref>; but using chemical transformation rather than electroporation). DNA fragments (143 bp or 306 bp) containing <italic>rpoB</italic> segments with GE-resistant or Rif-resistant mutations were prepared by PCR amplification using pRL706 derivatives carrying GE-resistant and Rif-resistant mutations as templates and 5’-CAGGTGGTATCCGTCGGTGCGTCCCTG-3’ and 5’-CGTTCCATACCAGTACCAACCAGCGGC-3’ (for GE-resistant mutations) or 5′-GGATATGATCAACGCCAAGCCGATTTCCGCAGC-3′ and 5′-CGATACGGAGTCTCAAGGAAGCCGTATTCG-3′ (for Rif-resistant mutations) as primers. DNA fragments were purified by isolation by electrophoresis on 0.8% agarose (procedures as in <xref ref-type="bibr" rid="bib57">Sambrook and Russell 2001</xref>) and extracted from gel slices using a Gel/PCR DNA Fragments Extraction Kit (IBI Scientific, Peosta, IA; procedures as specified by the manufacturer).</p><p>DNA fragments and co-selection/counter-selection plasmid pAKE604 (10 ng and 100 ng; for GE-resistant mutations) or DNA fragments only (30 ng; for Rif-resistant mutations) were introduced by transformation into chemically competent cells of <italic>E. coli</italic> D21f2tolC pKD46 (prepared by culturing <italic>E. coli</italic> D21f2tolC pKD46 in LB broth containing 200 μg/ml ampicillin and 1 mM arabinose at 30°C until OD = 0.6, pelleting cells, re-suspending cells in 85% LB, 10% PEG 3350, 5% DMSO, and 50 mM MgCl<sub>2</sub>, and flash freezing in dry-ice/ethanol), and transformants were cultured 3.5 hr at 37°C with shaking, applied to LB-agar plates containing 500 μg/ml GE and 40 μg/ml kanamycin (for GE-resistant mutations) or 1–2 μg/ml Rif (for Rif-resistant mutations), and incubated 24-30 hr at 37°C. Isolates containing chromosomal GE-resistant or Rif-resistant mutations were identified by the ability to form colonies on media containing GE or Rif, were confirmed by re-streaking on the same media, and were verified to have lost temperature-sensitive plasmid pKD46 by re-streaking on LB-agar plates containing 0 or 200 μg/ml ampicillin. For GE-resistant isolates, segregants lacking <italic>sacB</italic> plasmid pAKE604 were identified and verified by plating on LB agar containing 5% sucrose. Isolates were demonstrated to contain the expected mutations by PCR amplification and nucleotide sequencing of <italic>rpoB</italic>.</p></sec><sec id="s4-10"><title>GE-resistant mutants: determination of resistance levels</title><p>Resistance levels of GE-resistant mutants were quantified by performing broth microdilution assays. Single colonies were inoculated into 5 ml LB broth containing 200 μg/ml ampicillin, and 1 mM IPTG (for <italic>E. coli</italic> plasmid-borne mutants and controls), 5 ml LB broth (for <italic>E. coli</italic> chromosomal mutants and controls), or 5 ml TH broth (for <italic>S. pyogenes</italic> mutants and controls) and incubated at 37°C with shaking in air (for <italic>E. coli</italic>) or in 7% CO<sub>2</sub>/6% O<sub>2</sub>/4% H<sub>2</sub>/83% N<sub>2</sub> (for <italic>S. pyogenes</italic>) until OD<sub>600</sub> = 0.4–0.8. Diluted aliquots (∼4 × 10<sup>5</sup> cells in 50 μl of the same medium) were dispensed into wells of a 96-well plate containing 50 μl of the same medium or 50 μl of a twofold dilution series of GE in the same medium (final concentrations = 0 and 8–8000 μg/ml), and were incubated 16 hr at 37°C with shaking under the same conditions. The MIC was defined as the lowest tested concentration of GE that inhibited bacterial growth by ≥90%.</p></sec><sec id="s4-11"><title>GE-resistant mutants: determination of cross-resistance levels</title><p>Cross-resistance levels were determined analogously to resistance levels. Liquid cultures were prepared as described above for determination of resistance levels. Diluted aliquots of cultures (∼2 × 10<sup>5</sup> cells in 97 μl growth medium) were dispensed into wells of a 96-well plate, were supplemented with 3 μl methanol or 3 μl of a twofold dilution series of Rif, Sor, Stl, CBR703, Myx, or Lpm in methanol (final concentrations = 0 and 0.012–50 μg/ml), and were incubated 16 hr at 37°C with shaking.</p></sec><sec id="s4-12"><title>Formation of RNAP-promoter open complex</title><p>Reaction mixtures contained (20 μl): test compound (0 or 0.5 μM GE, or 2.2 μM Rif), 40 nM <italic>E. coli</italic> RNAP holoenzyme, 10 nM DNA fragment containing positions −42 to +426 of the <italic>lacUV5(ICAP)</italic> promoter (<xref ref-type="bibr" rid="bib50">Naryshkin et al., 2001</xref>), and 100 μg/ml heparin, in TB. Reaction components other than DNA and heparin were pre-incubated 10 min at 37°C; DNA was added and reaction mixtures were incubated 15 min at 37°C; heparin was added and reactions were incubated 2 min at 37°C to disrupt non-specific RNAP-promoter complexes and RNAP-promoter closed complexes (<xref ref-type="bibr" rid="bib8">Cech and McClure, 1980</xref>). Products were applied to 5% TBE polyacrylamide slab gels (Bio-Rad, Hercules, CA), gels were electrophoresed in TBE (90 mM Tris-borate, pH 8.0, and 2 mM EDTA), and gels were stained with SYBR Gold Nucleic Acid Gel Stain (Life Technologies).</p></sec><sec id="s4-13"><title>Nucleotide addition in transcription initiation: primer-dependent initiation</title><p>Reaction mixtures contained (20 μl): test compound (0 or 0.5 μM GE, or 2.2 μM Rif), 5 nM <italic>E. coli</italic> RNAP holoenzyme [Epicentre], 2.5 nM DNA fragment containing positions −49 to +30 of the <italic>lacCONS</italic> promoter (<xref ref-type="bibr" rid="bib47">Mukhopadhyay et al., 2001</xref>), 25 μg/ml heparin, 500 μM ApA, and 25 μM [α<sup>32</sup>P]UTP (0.9 Bq/fmol) in TB. Reaction components other than DNA, heparin, ApA, and [α-<sup>32</sup>P]UTP were pre-incubated 10 min at 37°C; DNA was added and reaction mixtures were incubated 15 min at 37°C; heparin was added and reaction mixtures were incubated 2 min at 37°C; ApA and [α<sup>32</sup>P]UTP were added and reaction mixtures were incubated 10 min at 37°C. Reactions were terminated by adding 10 μl 80% formamide, 10 mM EDTA, 0.04% bromophenol blue, 0.04% xylene cyanol, and 0.08% amaranth red. Products were heated 5 min at 90°C, cooled 5 min on ice, applied to 16% polyacrylamide (19:1 acrylamide:bisacrylamide, 7 M urea) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning (Typhoon; GE Healthcare, Piscataway, NJ). Identities of tri- and tetranucleotide abortive products from transcription initiation at lacUV5 were defined as in <xref ref-type="bibr" rid="bib4">Borowiec and Gralla (1985)</xref>.</p></sec><sec id="s4-14"><title>Nucleotide addition in transcription initiation: de novo initiation</title><p>Reaction mixtures contained (20 μl): 0 or 0.5 μM GE, 100 nM <italic>E. coli</italic> RNAP holoenzyme, 20 nM DNA fragment containing positions −65 to +35 of the bacteriophage T7 A1 promoter (prepared by PCR amplification of a synthetic nontemplate-strand oligodeoxyribonucleotide), 25 μg/ml heparin, 25 μM ATP, and 25 μM [α<sup>32</sup>P]UTP (0.7 Bq/fmol) in TB. Reaction components other than DNA, heparin, and NTPs were pre-incubated 5 min at 23°C, DNA was added and reaction mixtures were incubated 15 min at 37°C, heparin and NTPs were added were added and incubated 5 min at 37°C. Reactions were terminated by adding 10 μl 80% formamide, 10 mM EDTA, 0.04% bromophenol blue, and 0.04% xylene cyanol. Products were heated 5 min at 95°C, cooled 5 min on ice, and applied to 16% polyacrylamide (19:1 acrylamide:bisacrylamide, 7 M urea) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning (Typhoon; GE Healthcare).</p></sec><sec id="s4-15"><title>Nucleotide addition in transcription elongation: halted elongation complexes</title><p>Halted transcription elongation complexes (halted at position +29) were prepared essentially as in <xref ref-type="bibr" rid="bib54a">Revyakin et al. (2006)</xref>. Reaction mixtures (18 μl) contained: 40 nM <italic>E. coli</italic> RNAP holoenzyme, 10 nM DNA fragment N25-100-tR2 (<xref ref-type="bibr" rid="bib54a">Revyakin et al., 2006</xref>), 100 μg/ml heparin, 5 μM ATP, 5 μM GTP, and 5 μM [α<sup>32</sup>P]UTP (4 Bq/fmol) in TB. Reaction components except heparin and NTPs were pre-incubated 10 min at 37°C; heparin was added and reaction mixtures were incubated 2 min at 37°C; NTPs were added and reaction mixtures were incubated 5 min at 37°C. The resulting halted transcription elongation complexes were exposed to test compounds by addition of 1 μl 10 μM GE or 1 μl 44 μM Rif, incubated 5 min at 37°C, and were re-started by addition of 1 μl 1 mM CTP and incubation 5 min at 37°C. Reactions were terminated by adding 10 μl 80% formamide, 10 mM EDTA, 0.04% bromophenol blue, 0.04% xylene cyanol, and 0.08% amaranth red. Products were heated 5 min at 90°C, cooled 5 min on ice, applied to 16% polyacrylamide (19:1 acrylamide:bisacrylamide, 7 M urea) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning (Typhoon; GE Healthcare).</p></sec><sec id="s4-16"><title>Nucleotide addition in transcription elongation: reconstituted elongation complexes</title><p>Nucleic-acid scaffolds for assays were prepared as follows: nontemplate-strand oligodeoxyribonucleotide (5′-TCGCCAGACAGGG-3′; 1 μM), template-strand oligodeoxyribonucleotide (5′-CCCTGTCTGGCGATGGCGCGCCG-3′; 1 μM), and <sup>32</sup>P-5′-end-labelled oligoribonuceotide (5′-<sup>32</sup>P-CGGCGCGCC-3′; 1 μM; 200 Bq/fmol) in 25 μl 5 mM Tris–HCl, pH 7.7, 200 mM NaCl, and 10 mM MgCl<sub>2</sub>, were heated 5 min at 95°C and cooled to 4°C in 2°C steps with 1 min per step using a thermal cycler (Applied Biosystems) and then were stored at −20°C.</p><p>Reaction mixtures for assays contained (15 μl): 0 or 0.5 μM GE or 0 or 2.2 μM Rif, 40 nM wild-type <italic>E. coli</italic> RNAP core enzyme (Epicentre, Madison, WI), 10 nM <sup>32</sup>P-labelled nucleic-acid scaffold (200 Bq/fmol), and 20 μM ATP in TB. Reaction components except inhibitors and ATP were pre-incubated 5 min at 37°C, GE or Rif was added and reaction mixtures were incubated 5 min at 37°C, and ATP was added and reaction mixtures were incubated 2 min at 37°C. Reactions were terminated by adding 15 μl 80% formamide, 10 mM EDTA, 0.04% bromophenol blue, and 0.04% xylene cyanol, and heating 2 min at 95°C. Products were applied to 20% polyacrylamide (19:1 acrylamide:bisacrylamide, 7 M urea) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning (Typhoon; GE Healthcare).</p></sec><sec id="s4-17"><title>RNAP-Rif interaction assays</title><p>RNAP-Rif interaction assays were performed as in <xref ref-type="bibr" rid="bib20">Feklistov et al. (2008)</xref>. The assays monitored the quenching of the fluorescence emission of fluorescein incorporated into RNAP holoenzyme at σ<sup>70</sup> residue 517 (serving as a fluorescence-resonance-energy-transfer donor) by the naphthyl moiety of Rif (serving as a fluorescence-resonance-energy-transfer acceptor; <xref ref-type="bibr" rid="bib33">Knight et al., 2005</xref>; <xref ref-type="bibr" rid="bib20">Feklistov et al., 2008</xref>). Fluorescence measurements were performed using a QuantaMaster QM1 spectrofluorometer (PTI, Edison, NJ) (excitation wavelength = 480 nm; emission wavelength = 530 nm; and excitation and emission slit widths = 5 nm).</p><p>For determination of association kinetics, 720 μl 2 nM [F<sup>517</sup>]σ<sup>70</sup>-RNAP holoenzyme and 0-2 μM GE in 40 mM Tris–HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl<sub>2</sub>, 1 mM DTT, 0.02% Tween-20, and 5% glycerol was incubated 15 min at 24°C and then mixed with 30 μl 0.01–0.5 μM Rif in the same buffer at 24°C in a cuvette chamber with a mixing dead time ∼0.5 s, and fluorescence emission intensities were monitored for 30 min at 24°C. On-rates for RNAP-Rif interaction, k<sub>on</sub>, were calculated by fitting data to:<disp-formula id="equ1"><mml:math id="m1"><mml:mrow><mml:msub><mml:mrow><mml:mtext>I</mml:mtext><mml:mo>=</mml:mo><mml:mo>(</mml:mo><mml:mtext>I</mml:mtext></mml:mrow><mml:mn>0</mml:mn></mml:msub><mml:msub><mml:mrow><mml:mo>−</mml:mo><mml:mtext>I</mml:mtext></mml:mrow><mml:mi>∞</mml:mi></mml:msub><mml:msub><mml:mrow><mml:mo>)</mml:mo><mml:mtext>exp</mml:mtext><mml:mo>(</mml:mo><mml:mo>−</mml:mo><mml:mtext>k</mml:mtext></mml:mrow><mml:mrow><mml:mtext>obs</mml:mtext></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mtext>t</mml:mtext><mml:mo>)</mml:mo><mml:mo>+</mml:mo><mml:mtext>I</mml:mtext></mml:mrow><mml:mi>∞</mml:mi></mml:msub></mml:mrow></mml:math></disp-formula>where k<sub>obs</sub> is the observed association rate constant at a specified Rif concentration, I is the fluorescence emission intensity at time t, I<sub>o</sub> is the fluorescence emission intensity at t = 0, and I<sub>∞</sub> is the fluorescence emission intensity at t = ∞; followed by fitting the Rif-concentration-dependence of k<sub>obs</sub> to:<disp-formula id="equ2"><mml:math id="m2"><mml:mrow><mml:msub><mml:mtext>k</mml:mtext><mml:mrow><mml:mtext>obs</mml:mtext></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mo>=</mml:mo><mml:mtext>k</mml:mtext></mml:mrow><mml:mrow><mml:mtext>on</mml:mtext></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mtext>Rif</mml:mtext><mml:mo>]</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:mtext>k</mml:mtext></mml:mrow><mml:mrow><mml:mtext>off</mml:mtext></mml:mrow></mml:msub></mml:mrow></mml:math></disp-formula>where k<sub>off</sub> is ≥0 but is otherwise unconstrained.</p><p>For determination of dissociation kinetics, 720 μl of 2 nM [F<sup>517</sup>]σ<sup>70</sup>-RNAP holoenzyme and 0.05 μM Rif in the same buffer was incubated 30 min at 24°C and then mixed with 30 μl of 0–50 μM GE and 12.5–50 μM Sor (which binds to the same site as Rif but does not quench fluorescence emission and therefore serves as a ‘competitor trap’ for Rif dissociation kinetics; <xref ref-type="bibr" rid="bib20">Feklistov et al., 2008</xref>) in the same buffer at 24°C in a cuvette chamber with a mixing dead time ∼0.5 s; and fluorescence emission intensities were monitored for 5–300 min at 24°C. Dissociation kinetics were found not to depend on the concentration of Sor in the concentration range used in this work (final concentrations of 0.5–2 μM), verifying that Sor in this concentration range does not compete with GE and does not actively displace Rif from RNAP. Off-rates for RNAP-Rif interaction, k<sub>off</sub>, were calculated as:<disp-formula id="equ3"><mml:math id="m3"><mml:mrow><mml:msub><mml:mrow><mml:mtext>I</mml:mtext><mml:mo>=</mml:mo><mml:mtext>I</mml:mtext></mml:mrow><mml:mn>0</mml:mn></mml:msub><mml:msub><mml:mrow><mml:mo>+</mml:mo><mml:mo>(</mml:mo><mml:mtext>I</mml:mtext></mml:mrow><mml:mi>∞</mml:mi></mml:msub><mml:msub><mml:mrow><mml:mo>−</mml:mo><mml:mtext>I</mml:mtext></mml:mrow><mml:mtext>0</mml:mtext></mml:msub><mml:msub><mml:mrow><mml:mo>)</mml:mo><mml:mo>[</mml:mo><mml:mtext>1</mml:mtext><mml:mo>−</mml:mo><mml:mtext>exp</mml:mtext><mml:mo>(</mml:mo><mml:mo>−</mml:mo><mml:mtext>k</mml:mtext></mml:mrow><mml:mrow><mml:mtext>off</mml:mtext></mml:mrow></mml:msub><mml:mtext>t</mml:mtext><mml:mo>)</mml:mo><mml:mo>]</mml:mo></mml:mrow></mml:math></disp-formula>where I is the fluorescence emission intensity at time t, I<sub>o</sub> is the fluorescence intensity at t = 0, and I<sub>∞</sub> is the fluorescence intensity at t = ∞.</p><p>Equilibrium dissociation constants for RNAP-Rif interaction, K<sub>d</sub>, were calculated as k<sub>off</sub>/k<sub>on</sub>.</p><p>The equilibrium dissociation constant for RNAP-GE interaction, K<sub>i</sub>, was calculated from the association-kinetics data, by fitting the GE-concentration-dependence of I<sub>∞</sub> to:<disp-formula id="equ4"><mml:math id="m4"><mml:mrow><mml:msub><mml:mtext>I</mml:mtext><mml:mi>∞</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mtext>I</mml:mtext></mml:mrow><mml:mrow><mml:mi>∞</mml:mi><mml:mtext>,max</mml:mtext></mml:mrow></mml:msub><mml:mo>[</mml:mo><mml:mtext>GE</mml:mtext><mml:mo>]</mml:mo><mml:mo>)</mml:mo></mml:mrow><mml:mo>/</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mtext>K</mml:mtext></mml:mrow><mml:mtext>i</mml:mtext></mml:msub><mml:mo>+</mml:mo><mml:mo>[</mml:mo><mml:mtext>GE</mml:mtext><mml:mo>]</mml:mo><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:math></disp-formula></p></sec><sec id="s4-18"><title>Structure determination: RNAP + GE</title><p>Crystallization and crystal handling were performed essentially as in <xref ref-type="bibr" rid="bib75">Tuske et al. (2005)</xref>. A crystallization stock solution was prepared by adding 1 μl <italic>T. thermophilus</italic> RNAP holoenzyme (10 mg/ml) in 20 mM Tris–HCl, pH 7.7, 100 mM NaCl, and 1% glycerol to 1 μl 33 mM magnesium formate containing 40 μM ZnCl<sub>2</sub>. The crystallization stock solution was equilibrated against a reservoir solution of 30 mM sodium citrate, pH 5.4, and 35 mM magnesium formate in a vapor-diffusion hanging-drop crystallization tray (Hampton Research, Aliso Viejo, CA) at 22°C. Hexagonal crystals formed and grew to a final size of ∼0.4 × ∼0.4 × ∼0.2 mm within 6 d.</p><p>GE was soaked into RNAP crystals by addition of 0.2 μl 10 mM GE in 60% (vol/vol) (±)-2-methyl-2,4-pentanediol (MPD; Hampton Research) to the crystallization drop and incubation 15 min at 22°C. Crystals were transferred to solutions containing 0.5 mM GE, 20 mM MES, pH 6.0, 13 mM magnesium formate, 2 mM spermine, 2 mM DTT, 5% PEG400, and 15% (vol/vol) (2R,3R)-(−)-2,3-butanediol (Sigma–Aldrich), and were flash-cooled with liquid nitrogen.</p><p>Diffraction data for RNAP-GE were collected at Cornell High Energy Synchrotron Source (CHESS) beamline F1 and were processed and scaled using iMOSFLM and SCALA (<xref ref-type="bibr" rid="bib3">Battye et al., 2011</xref>; <xref ref-type="bibr" rid="bib19">Evans, 2006</xref>). The structure of RNAP-GE was solved by molecular replacement with AutoMR in Phenix (<xref ref-type="bibr" rid="bib45">McCoy et al., 2007</xref>) using a modified structure of <italic>T. thermophilus</italic> RNAP holoenzyme (PDB 3DXJ; <xref ref-type="bibr" rid="bib46">Mukhopadhyay et al., 2008</xref>) as the search model. Early stages of refinement of the RNAP-GE complex included rigid-body refinement of subdomains (∼15–200 residue segments) of the RNAP molecule. Cycles of rigid-body, individual-atom, and individual-B-factor refinement using Ramachandran and secondary structure restraints and optimized weights for stereochemistry and optimized atomic displacement parameters were carried out using Phenix (<xref ref-type="bibr" rid="bib1">Adams et al., 2010</xref>). Manual rebuilds against electron-density maps were performed using Coot (<xref ref-type="bibr" rid="bib18">Emsley et al., 2010</xref>) and Molprobity (<xref ref-type="bibr" rid="bib14">Davis et al., 2007</xref>; <xref ref-type="bibr" rid="bib9">Chen et al., 2010</xref>). In addition, two refinement cycles were performed within Autobuster (<xref ref-type="bibr" rid="bib5">Bricogne et al., 2011</xref>). For GE backbone atoms and GE sidechain atoms with previously defined stereochemistry (<xref ref-type="bibr" rid="bib40">Marazzi et al., 2005</xref>), an initial atomic model was generated using Maestro (Schrodinger, Portland, OR) and was fit to mFo-DFc maps using Phenix (<xref ref-type="bibr" rid="bib1">Adams et al., 2010</xref>). For GE sidechain atoms with previously undefined stereochemistry, stereochemistry was deduced and atoms were added based on assessment of mFo-DFc maps and RNAP-GE interactions using PrimeX (Schrodinger). All GE atoms could be fitted to density except atoms of the GE dmaDap residue distal to the sidechain carbonyl moiety. Subsequent cycles of refinement and model building were performed, leading to the current crystallographic model, with a standard crystallographic residual of R<sub>work</sub> = 0.21 and R<sub>free</sub> = 0.24 computed using all data from 38.97 to 3.35 Å resolution. Atomic coordinates and structure factors for RNAP-GE have been deposited in the PDB with accession code 4MQ9.</p></sec><sec id="s4-19"><title>Structure determination: RP<sub>o</sub> + GE</title><p>Crystals of <italic>T. thermophilus</italic> RP<sub>o</sub> were prepared using the same nucleic-acid scaffold as used for analysis of RP<sub>o</sub> in <xref ref-type="bibr" rid="bib84">Zhang et al. (2012)</xref>, and were grown and handled essentially as in <xref ref-type="bibr" rid="bib84">Zhang et al. (2012)</xref>. Crystallization drops contained 1 μl RP<sub>o</sub> in 20 mM Tris–HCl, pH 7.7, 100 mM NaCl, and 1% glycerol, and 1 μl reservoir buffer (RB; 100 mM Tris–HCl, pH 8.4, 200 mM KCl, 50 mM MgCl<sub>2</sub>, and 9.5% PEG4000), and were equilibrated against 400 μl RB in a vapor-diffusion hanging-drop tray. Rod-like crystals appeared in 1 d, and were used to micro-seed hanging drops using the same conditions.</p><p>GE was soaked into RP<sub>o</sub> crystals by addition of 0.2 μl 20 mM GE in RB to the crystallization drop and incubation 15 min at 22°C. Crystals were transferred in stepwise fashion to successive reservoir solutions containing 1 mM GE in 0.5%, 1%, 2.5%, 5%, 10%, 14%, and 17.5% (v/v) (2R, 3R)-(−)-2,3-butanediol (20 s for first step and 2 s for each subsequent step) and were flash-cooled with liquid nitrogen.</p><p>Diffraction data were collected at CHESS beamline F1 and Brookhaven National Laboratory (BNL) beamline X29A and were processed using HKL2000 (<xref ref-type="bibr" rid="bib52">Otwinowski and Minor, 1997</xref>). Structure factors were converted using the French-Wilson algorithm in Phenix (<xref ref-type="bibr" rid="bib21">French and Wilson, 1978</xref>) and were subjected to anisotropy correction using the UCLA MBI Diffraction Anisotropy server (<xref ref-type="bibr" rid="bib71">Strong et al., 2006</xref>; <ext-link ext-link-type="uri" xlink:href="http://services.mbi.ucla.edu/anisoscale/">http://services.mbi.ucla.edu/anisoscale/</ext-link>). The structure was solved by molecular replacement with Molrep (<xref ref-type="bibr" rid="bib76">Vagin and Teplyakov, 1997</xref>) using one RNAP molecule from the structure of <italic>T. thermophilus</italic> RP<sub>o</sub> (PDB 4 G7H; <xref ref-type="bibr" rid="bib84">Zhang et al., 2012</xref>) as the search model. Early-stage refinement included rigid-body refinement of the RNAP molecule, followed by rigid-body refinement of each subunit of RNAP molecule. Cycles of iterative model building with Coot (<xref ref-type="bibr" rid="bib18">Emsley et al., 2010</xref>) and refinement with Phenix (<xref ref-type="bibr" rid="bib1">Adams et al., 2010</xref>) were performed. Atomic models of the DNA nontemplate strand, the DNA template strand, and GE were built into mFo-DFc omit maps, and subsequent cycles of refinement and model building were performed. The final crystallographic model of RP<sub>o</sub>-GE, refined to R<sub>work</sub> and R<sub>free</sub> of 0.21 and 0.25, has been deposited in the PDB with accession code 4OIN.</p></sec><sec id="s4-20"><title>Structure determination: RP<sub>o</sub> + ATP + CMPcPP</title><p>ATP (Sigma–Aldrich) and CMPcPP (Jena Biosciences, Jena, Germany) were soaked into RP<sub>o</sub> crystals (prepared as described above, using the nucleic-acid scaffold used for analysis of RP<sub>o</sub>-GpA in <xref ref-type="bibr" rid="bib84">Zhang et al. 2012</xref>) by addition of 0.2 μl 30 mM ATP and 30 mM CMPcPP in 55% (vol/vol) RB to the crystallization drop, and incubation 15–20 min at 22°C. Crystals were transferred into reservoir solutions containing 2 mM ATP and 2 mM CMPcPP in 17.5% (vol/vol) (2R, 3R)-(−)-2,3-butanediol and were flash-cooled with liquid nitrogen.</p><p>Diffraction data were collected at BNL beamline X25, processed and scaled using HKL2000 (<xref ref-type="bibr" rid="bib52">Otwinowski and Minor, 1997</xref>), and subjected to anisotropic correction using the UCLA MBI Diffraction Anisotropy server (<xref ref-type="bibr" rid="bib71">Strong et al., 2006</xref>; <ext-link ext-link-type="uri" xlink:href="http://services.mbi.ucla.edu/anisoscale/">http://services.mbi.ucla.edu/anisoscale/</ext-link>). The structure was solved and refined using procedures analogous to those described above for RP<sub>o</sub>-GE. The final crystallographic model contained RP<sub>o</sub>, ATP bound in the RNAP i site, and CMPcPP:Mg<sup>2+</sup> bound in the RNAP i+1 site. The final crystallographic model of RP<sub>o</sub>-ATP-CMPcPP, refined to R<sub>work</sub> and R<sub>free</sub> of 0.21 and 0.26, respectively, has been deposited in the PDB with accession code 4OIO.</p></sec><sec id="s4-21"><title>Structure determination: RP<sub>o</sub> + GE + ATP + CMPcPP</title><p>Crystals of RP<sub>o</sub> (prepared as described above for RP<sub>o</sub> + ATP + CMPcPP) first were soaked with GE (addition of 0.2 μl 20 mM GE in RB to the crystallization drop and incubation 15 min at 22°C) and then were soaked with ATP and CMPcPP (addition of 0.2 μl 30 mM ATP and 30 mM CMPcPP in 55% [vol/vol] RB to the crystallization drop and incubation 15 min at 22°C). Crystals then were transferred to reservoir solutions containing 1 mM GE, 2 mM ATP, and 2 mM CMPcPP in 17.5% (vol/vol) (2R, 3R)-(−)-2,3-butanediol and were flash-cooled with liquid nitrogen.</p><p>Diffraction data were collected at BNL beamline X25, and were processed, scaled, and corrected for anisotropy using HKL2000 (<xref ref-type="bibr" rid="bib52">Otwinowski and Minor, 1997</xref>). The structure was solved and refined using procedures analogous to those described above for RP<sub>o</sub>-GE. The final crystallographic model contained RP<sub>o,</sub> GE bound to the GE target, and ATP:Mg<sup>2+</sup> bound to the RNAP E site, and did not contain ATP in the RNAP i site or CMPcPP in RNAP i+1 site. The final crystallographic model, refined to R<sub>work</sub> and R<sub>free</sub> of 0.21 0.25, respectively, has been deposited in the PDB with accession code 4OIP.</p></sec><sec id="s4-22"><title>Structure determination: RP<sub>o</sub> + GE + Rif</title><p>Crystals of RP<sub>o</sub> (prepared as described above for RP<sub>o</sub> + ATP + CMPcPP) first were soaked with Rif (addition of 0.1 μl 20 mM Rif in RB containing 40% [vol/vol] [2R, 3R]-(−)-2,3-butanediol to the crystallization drop and incubation 15 min at 22°C) and then were soaked with GE (addition of 0.2 μl 20 mM GE in RB to the crystallization drop and incubation 15 min at 22°C). Crystals then were transferred to reservoir solutions containing 1 mM GE and 0.4 mM Rif in 17.5% (vol/vol) (2R, 3R)-(−)-2,3-butanediol and were flash-cooled with liquid nitrogen.</p><p>Diffraction data were collected at CHESS beamline F1, and were processed and scaled using HKL2000 (<xref ref-type="bibr" rid="bib52">Otwinowski and Minor, 1997</xref>). The structure was solved and refined using procedures analogous to those described above for RP<sub>o</sub>-GE. The final crystallographic model contained RP<sub>o</sub> and GE bound to the GE target but did not contain Rif. The final crystallographic model, refined to R<sub>work</sub> and R<sub>free</sub> of 0.20 and 0.25, respectively, has been deposited in the PDB with accession code 4OIQ.</p></sec><sec id="s4-23"><title>Structure determination: RP<sub>o</sub> + GE + RifSV</title><p>Crystals of RPo (prepared as described above for RP<sub>o</sub> + ATP + CMPcPP) first were soaked with RifSV (addition of 0.2 μl 10 mM RifSV in RB to the crystallization drop and incubation 15 min at 22°C, or transfer of the crystal to 1 μl 10 mM RifSV in RB and incubation 15 min at 22°C) and then were soaked with GE (addition of 0.2 μl 20 mM GE in RB to the drop and incubation 15 min at 22°C). Crystals then were transferred in to reservoir solutions containing 1 mM GE and 1 mM RifSV in 17.5% (vol/vol) (2R, 3R)-(−)-2,3-butanediol and were flash-cooled with liquid nitrogen.</p><p>Diffraction data were collected at BNL beamline X25, were processed and scaled using HKL2000 (<xref ref-type="bibr" rid="bib52">Otwinowski and Minor, 1997</xref>), and were subjected to anisotropic correction using the UCLA MBI Diffraction Anisotropy server (<xref ref-type="bibr" rid="bib71">Strong et al., 2006</xref>; <ext-link ext-link-type="uri" xlink:href="http://services.mbi.ucla.edu/anisoscale/">http://services.mbi.ucla.edu/anisoscale/</ext-link>). The structure was solved and refined using procedures analogous to those described above for RP<sub>o</sub>-GE. The final crystallographic model contained RP<sub>o</sub>, GE bound to the GE target, and RifSV bound to the Rif target. The final crystallographic model of RP<sub>o</sub>-GE-RifSV, refined to R<sub>work</sub> and R<sub>free</sub> of 0.21 and 0.25, respectively, has been deposited in the PDB with accession code 4OIR.</p></sec><sec id="s4-24"><title>Synthesis of a GE-rifamycin bipartite inhibitor: step 1, synthesis of [ζ<sup>1</sup>-amino-dmaDap; α-descarboxy-Ama]GE (compound 1 of <xref ref-type="fig" rid="fig7">Figure 7C</xref>)</title><p><fig id="fig8" position="float"><graphic xlink:href="elife02450f008"/></fig></p><p>GE (20 mg 25 μmol), ammonium acetate (60 mg; 780 μmol; Aldrich), and perchloric-acid-impregnated silica (5 mg; prepared as in <xref ref-type="bibr" rid="bib67">Singh et al., 2009</xref>), were mixed in 4 ml absolute ethanol in a screw-cap vial. The mixture was microwaved for 4 × 30 s (1000 W) with intervals of 1 min for re-mixing contents of the vial. The mixture was allowed to incubate at room temperature for another 16 hr, evaporated to dryness, and resuspended in 2 ml 1% triethylamine-water. The mixture was centrifuged, and the supernatant was purified via HPLC (Phenomenex C18, semi-prep; 5 min 0% B, 20 min 5% B, 25 min 10% B, 30 min 30% B, 40 min 80% B; A = water, B = acetonitrile, 2 ml/min).</p><p>The HPLC elution profile and mass spectrum of the product indicate that the product has undergone decarboxylation of the Ama sidechain (<xref ref-type="bibr" rid="bib42">Mariani et al., 2005</xref>). It is known that acid and heat induce decarboxylation of the GE Ama sidechain, and that decarboxylated GE exhibits ∼1/20 the RNAP-inhibitory activity and antibacterial activity of GE (<xref ref-type="bibr" rid="bib42">Mariani et al., 2005</xref>).</p><p>Yield: 3.5 mg; 18%.</p><p>MS (MALDI): calculated: <italic>m/z</italic> 777.80 (MH<sup>+</sup>); found: 778.20, 800.59 (M + Na<sup>+</sup>).</p></sec><sec id="s4-25"><title>Synthesis of GE-rifamycin bipartite inhibitor: step 2, synthesis of {[α-descarboxy-Ama]GE}-NH-{rifamycin S} (compound 2 of <xref ref-type="fig" rid="fig7">Figure 7C</xref>)</title><p><fig id="fig9" position="float"><graphic xlink:href="elife02450f009"/></fig></p><p>3-Bromo-rifamycin S (2.7 mg; 3.47 μmol; prepared as in <xref ref-type="bibr" rid="bib41">Marchi and Montecchi 1979</xref>), compound 1 (2.7 mg; 3.47 μmol; Example 1a) and triethylamine (0.5 μl; 3.47 μmol; Aldrich) were mixed together in 200 μl DMF and allowed to react for 18 hr at 25°C. The reaction mixture was quenched with 100 μl water, centrifuged, and the supernatant was purified via HPLC (Phenomenex C18, semi-prep; 0 min 10% B, 35 min 100% B; A = water, B = acetonitrile, 2 ml/min).</p><p>Yield: 1.51 mg; 30%.</p><p>MS (MALDI): calculated: <italic>m/z</italic> 1493.52 (M + Na<sup>+</sup>); found: 1494.22.</p></sec><sec id="s4-26"><title>Synthesis of GE-rifamycin bipartite inhibitor: step 3, synthesis of {[α-descarboxy-Ama]GE}-NH-{RifSV} (compound 3 of <xref ref-type="fig" rid="fig7">Figure 7C</xref>; “RifaGE-3”)</title><p><fig id="fig10" position="float"><graphic xlink:href="elife02450f010"/></fig></p><p>Sodium ascorbate (2.38 mg; 12 μmol; Aldrich) in 25 μl water was added to compound 2 (0.600 mg; 0.4 μmol; Example 1b) in 100 μl water, mixed, and allowed to react for 10 min at 25°C. The product was isolated via HPLC (Phenomenex C18, analytical; 0’ 10% B, 35′ 100% B; A = water, B = acetonitrile, 1 ml/min).</p><p>Yield: 0.1 mg; 17%.</p><p>MS (MALDI): calculated: m/z 1495.52 (M + Na+); found: 1495.71.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank the Brookhaven National Synchrotron Light Source and the Cornell High Energy Synchrotron Source for beamline access for X-ray data collection, and M Chinnaraj, Q Jiang, S Ismail, S Liu, S Mandal, C O’Brien, A Srivastava, and X Wang for assistance.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>YZ: patent pending on bipartite inhibitors.</p></fn><fn fn-type="conflict" id="conf2"><p>YWE: patent pending on bipartite inhibitors.</p></fn><fn fn-type="conflict" id="conf3"><p>DD: license distributions from Merck &amp; Co.; patent pending on bipartite inhibitors.</p></fn><fn fn-type="conflict" id="conf4"><p>RHE: license distributions from Merck &amp; Co.; patent pending on bipartite inhibitors.</p></fn><fn fn-type="conflict" id="conf5"><p>KYE: license distributions from Merck &amp; Co.</p></fn><fn fn-type="conflict" id="conf6"><p>SM: Employee and shareholder of Naicons Srl.</p></fn><fn fn-type="conflict" id="conf7"><p>SD: Employee and shareholder of Naicons Srl.</p></fn><fn fn-type="conflict" id="conf8"><p>The other authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>YZ, Approved version to be published, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con2"><p>DD, Approved version to be published, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>MXH, Approved version to be published, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>ES, Approved version to be published, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>KYE, Approved version to be published, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>YWE, Approved version to be published, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>VM, Approved version to be published, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con8"><p>HV-M, Approved version to be published, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con9"><p>YF, Approved version to be published, Acquisition of data</p></fn><fn fn-type="con" id="con10"><p>RY, Approved version to be published, Acquisition of data</p></fn><fn fn-type="con" id="con11"><p>ST, Approved version to be published, Acquisition of data</p></fn><fn fn-type="con" id="con12"><p>HI, Approved version to be published, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con13"><p>RJ, Approved version to be published, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con14"><p>SM, Approved version to be published, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con15"><p>SD, Approved version to be published, Analysis and interpretation of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con16"><p>EA, Approved version to be published, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con17"><p>RHE, Approved version to be published, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.02450.021</object-id><label>Supplementary file 1.</label><caption><p>(<bold>A</bold>) GE: antibacterial activity. (<bold>B</bold>) GE: RNAP-inhibitory activity.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.021">http://dx.doi.org/10.7554/eLife.02450.021</ext-link></p></caption><media mime-subtype="doc" mimetype="application" xlink:href="elife02450s001.doc"/></supplementary-material><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.02450.022</object-id><label>Supplementary file 2.</label><caption><p>(<bold>A</bold>) ‘Doped’ oligonucleotide primers used for saturation mutagenesis. (<bold>B</bold>) Chromosomal GE<sup>R</sup> mutants in <italic>E. coli</italic> D21f2tolC: sequences and properties. (<bold>C</bold>) Chromosomal GE<sup>R</sup> mutants in <italic>S. pyogenes</italic>: sequences and properties. (<bold>D</bold>) Chromosomal GE<sup>R</sup> mutants in <italic>E. coli</italic> D21f2tolC: absence of cross-resistance to Rif. (<bold>E</bold>) Chromosomal Rif<sup>R</sup> mutants in <italic>E. coli</italic> D21f2tolC: absence of cross-resistance to GE. (<bold>F</bold>) GE<sup>R</sup> mutants from saturation mutagenesis: absence of cross-resistance to Sor, Stl, CBR703, Myx, and Lpm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02450.022">http://dx.doi.org/10.7554/eLife.02450.022</ext-link></p></caption><media mime-subtype="doc" mimetype="application" xlink:href="elife02450s002.doc"/></supplementary-material><sec sec-type="datasets"><title>Major datasets</title><p>The following datasets were generated:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Ho</surname><given-names>MX</given-names></name>, <name><surname>Arnold</surname><given-names>E</given-names></name>, <name><surname>Ebright</surname><given-names>RH</given-names></name>, <name><surname>Zhang</surname><given-names>Y</given-names></name>, and <name><surname>Tuske</surname><given-names>S</given-names></name>, <year>2013</year><x>, </x><source>Crystal structure of <italic>Thermus thermophilus</italic> RNA polymerase holoenzyme in complex with GE23077</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4MQ9">http://www.rcsb.org/pdb/explore/explore.do?structureId=4MQ9</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro2"><name><surname>Zhang</surname><given-names>Y</given-names></name>, <name><surname>Arnold</surname><given-names>E</given-names></name>, and <name><surname>Ebright</surname><given-names>RH</given-names></name>, <year>2014</year><x>, </x><source>Crystal structure of <italic>Thermus thermophilus</italic> transcription initiation complex soaked with GE23077</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIN">http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIN</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id=" Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro3"><name><surname>Zhang</surname><given-names>Y</given-names></name>, <name><surname>Arnold</surname><given-names>E</given-names></name>, and <name><surname>Ebright</surname><given-names>RH</given-names></name>, <year>2014</year><x>, </x><source>Crystal structure of <italic>Thermus thermophilus</italic> pre-insertion substrate complex for de novo transcription initiation</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIO">http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIO</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro4"><name><surname>Zhang</surname><given-names>Y</given-names></name>, <name><surname>Arnold</surname><given-names>E</given-names></name>, and <name><surname>Ebright</surname><given-names>RH</given-names></name>, <year>2014</year><x>, </x><source>Crystal structure of <italic>Thermus thermophilus</italic> transcription initiation complex soaked with GE23077, ATP, and CMPcPP</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIP">http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIP</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro5"><name><surname>Zhang</surname><given-names>Y</given-names></name>, <name><surname>Arnold</surname><given-names>E</given-names></name>, and <name><surname>Ebright</surname><given-names>RH</given-names></name>, <year>2014</year><x>, </x><source>Crystal structure of <italic>Thermus thermophilus</italic> transcription initiation complex soaked with GE23077 and rifampicin</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIQ">http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIQ</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro6"><name><surname>Zhang</surname><given-names>Y</given-names></name>, <name><surname>Arnold</surname><given-names>E</given-names></name>, and <name><surname>Ebright</surname><given-names>RH</given-names></name>, <year>2014</year><x>, </x><source>Crystal structure of <italic>Thermus thermophilus</italic> RNA polymerase transcription initiation complex soaked with GE23077 and rifamycin SV</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIR">http://www.rcsb.org/pdb/explore/explore.do?structureId=4OIR</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank.</comment></related-object></p><p><bold>Reporting standards:</bold> Standard used to collect data: <ext-link ext-link-type="uri" xlink:href="http://www.wwpdb.org/policy.html">http://www.wwpdb.org/policy.html</ext-link>.</p></sec></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group 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