Title: Mutations of Bacterial RNA Polymerase Leading to Resistance to Microcin J25
Abstract: A mutation in the conserved segment of therpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, β′, was found to make Escherichia colicells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M. A., Rintoul, M. R., Farias, R. N., and Salomon, R. A. (2001) J. Bacteriol. 183, 4543–4550). Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25. We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro. The new mutations affect β′ amino acids in evolutionarily conserved segments G, G′, and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP β subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center. A mutation in the conserved segment of therpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, β′, was found to make Escherichia colicells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M. A., Rintoul, M. R., Farias, R. N., and Salomon, R. A. (2001) J. Bacteriol. 183, 4543–4550). Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25. We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro. The new mutations affect β′ amino acids in evolutionarily conserved segments G, G′, and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP β subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center. RNA polymerase streptolydigin microcin J25 isopropyl-1-thio-β-d-galactopyranoside Bacterial RNA polymerase (RNAP)1 is the central enzyme of gene expression and a target of genetic regulation. The catalytically proficient core enzyme is composed of five polypeptides: the largest subunit β′, the second largest subunit β, the dimer of identical α subunits, and a small subunit ω. Upon the binding of one of the several ς specificity subunits the core is converted to a holoenzyme that can specifically initiate transcription from promoters. RNAP is a target of several inhibitors. Interest is attached to these low molecular weight compounds as they can be used as tools to reveal new information about RNAP mechanism and can also be used as antibacterial drugs. The best studied bacterial RNAP inhibitor, rifampicin, is widely used against mycobacterial infections. Rifampicin and its derivatives bind to the RNAP β subunit (1Ovchinnikov Y.A. Monastyrskaya G.S. Guriev S.O. Kalinina N.F. Sverdlov E.D. Gragerov A.I. Bass I.A. Kiver I.F. Moiseyeva E.P. Igumnov V.N. Mindlin S.Z. Nikiforov V.G. Khesin R.B. Mol. Gen. Genet. 1983; 190: 344-348Crossref PubMed Scopus (80) Google Scholar, 2Jin D.J. Gross C.A. J. Mol. Biol. 1988; 202: 45-58Crossref PubMed Scopus (545) Google Scholar, 3Severinov K. Soushko M. Goldfarb A. Nikiforov V. J. Biol. Chem. 1993; 268: 14280-14825Abstract Full Text PDF Google Scholar, 4Campbell E.A. Korzheva N. Mustaev A. Murakami K. Nair S. Goldfarb A. Darst S.A. Cell. 2001; 104: 901-912Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar) and prevent synthesis of RNAs longer than two-three nucleotides in length by occluding the nascent RNA exit path (5McClure W.R. Cech C.L. J. Biol. Chem. 1978; 253: 8949-8956Abstract Full Text PDF PubMed Google Scholar, 6Schulz W. Zillig W. Nucleic Acids Res. 1981; 9: 6889-6906Crossref PubMed Scopus (48) Google Scholar). An unrelated inhibitor, streptolydigin (Stl), either affects the binding of incoming NTP in the substrate binding site of RNAP or directly targets the catalysis of phosphodiester bond formation (7Cassani G. Burgess R.R. Goodman H.M. Gold L. Nat. New Biol. 1971; 230: 197-200Crossref PubMed Scopus (62) Google Scholar, 8Helm K.v. d. Krakow J.S. Nat. New Biol. 1972; 235: 82-83Crossref PubMed Scopus (21) Google Scholar, 9McClure W.R. J. Biol. Chem. 1980; 255: 1610-1616Abstract Full Text PDF PubMed Google Scholar). Mutations causing RNAP resistance to Stl were mapped to both the rpoB and rpoCgenes, coding for the β and β′ subunits, respectively (3Severinov K. Soushko M. Goldfarb A. Nikiforov V. J. Biol. Chem. 1993; 268: 14280-14825Abstract Full Text PDF Google Scholar, 10Lisitsyn N.A. Sverdlov E.D. Moiseyeva E.P. Nikiforov V.G. Bioorg. Khim. 1985; 11: 132-134PubMed Google Scholar, 11Heisler L.M. Suzuki H. Landick R. Gross C.A. J. Biol. Chem. 1993; 268: 25369-25375Abstract Full Text PDF PubMed Google Scholar, 12Severinov K. Markov D. Nikiforov V. Severinova E. Landick R. Darst S.A. Goldfarb A. J. Biol. Chem. 1995; 270: 23926-23929Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The β′ site where Stl-resistant substitutions were localized overlaps the site in eukaryal RNAP II largest subunit where substitutions leading to resistance to α-amanitin, a peptide that specifically inhibits PNAP II transcription, map (7Cassani G. Burgess R.R. Goodman H.M. Gold L. Nat. New Biol. 1971; 230: 197-200Crossref PubMed Scopus (62) Google Scholar, 8Helm K.v. d. Krakow J.S. Nat. New Biol. 1972; 235: 82-83Crossref PubMed Scopus (21) Google Scholar, 12Severinov K. Markov D. Nikiforov V. Severinova E. Landick R. Darst S.A. Goldfarb A. J. Biol. Chem. 1995; 270: 23926-23929Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 13Bartolomei M. Corden J. Mol. Cell. Biol. 1987; 7: 586-594Crossref PubMed Scopus (74) Google Scholar, 14Bartolomei M.S. Corden J.L. Mol. Gen. Genet. 1995; 246: 778-782Crossref PubMed Scopus (28) Google Scholar, 15Allison L.A. Moyle M. Shales M. Ingles C.J. Cell. 1985; 42: 599-610Abstract Full Text PDF PubMed Scopus (444) Google Scholar). Thus, Stl and α-amanitin may inhibit transcription in their respective systems through similar mechanisms, despite the lack of chemical similarity between the two drugs. The third antibiotic known to target bacterial RNAP, tagetitoxin, inhibits transcription by slowing the rate of RNAP elongation and promoting pausing (16Mathews D.E. Durbin R.D. J. Biol. Chem. 1990; 265: 493-498Abstract Full Text PDF PubMed Google Scholar). The site of RNAP that tagetitoxin interacts with is not known. Tagetitoxin also inhibits transcription by eukaryotic RNAP III (17Steinberg T.H. Mathews D.E. Durbin R.D. Burgess R.R. J. Biol. Chem. 1990; 265: 499-505Abstract Full Text PDF PubMed Google Scholar), indicating that the tagetitoxin binding site may be evolutionarily conserved. Recently, one of our groups (18Delgado M.A. Rintoul M.R. Farias R.N. Salomon R.A. J. Bacteriol. 2001; 183: 4543-4550Crossref PubMed Scopus (139) Google Scholar) reported that Escherichia coli cells harboring a mutation in the rpoC gene, which codes for RNAP β′, became resistant to microcin J25 (18Delgado M.A. Rintoul M.R. Farias R.N. Salomon R.A. J. Bacteriol. 2001; 183: 4543-4550Crossref PubMed Scopus (139) Google Scholar). Microcin J25 (MccJ25) is a bactericidal peptide made of 21 amino acids (19Salomon R.A. Farias R.N. J. Bacteriol. 1992; 174: 7428-7435Crossref PubMed Google Scholar, 20Blond A. Cheminant M. Segalas-Milazzo I. Peduzzi J. Barthelemy M. Goulard C. Salomon R. Moreno F. Farias R. Rebuffat S. Eur. J. Biochem. 2001; 268: 2124-2133Crossref PubMed Scopus (38) Google Scholar). MccJ25-producing cells harbor a plasmid that is responsible for MccJ25 production and resistance of MccJ25-producing cells to the drug (21Solbiati J.O. Ciaccio M. Farias R.N. Salomon R.A. J. Bacteriol. 1996; 178: 3661-3663Crossref PubMed Google Scholar). MccJ25 production increases when cells reach stationary phase and nutrients become limiting, thus giving MccJ25-producing cells an advantage (19Salomon R.A. Farias R.N. J. Bacteriol. 1992; 174: 7428-7435Crossref PubMed Google Scholar, 22Chiuchiolo M.J. Delgado M.A. Farias R.N. Salomon R.A. J. Bacteriol. 2001; 183: 1755-1764Crossref PubMed Scopus (41) Google Scholar). Most of spontaneous MccJ25-resistant mutants affect genes encoding cytoplasmic membrane proteins and appear to be intake mutants (23Salomon R.A. Farias R.N. J. Bacteriol. 1995; 177: 3323-3325Crossref PubMed Google Scholar). The fact that a rare microcin resistance mutation resulted in altered RNAP suggested that RNAP may be the cellular target of MccJ25. In agreement with this idea, it has been shown that in vitro activity ofE. coli RNAP is reduced in the presence of micromolar concentrations of MccJ25 (18Delgado M.A. Rintoul M.R. Farias R.N. Salomon R.A. J. Bacteriol. 2001; 183: 4543-4550Crossref PubMed Scopus (139) Google Scholar). The rpoC mutation that resulted in MccJ25 resistance caused a substitution of an evolutionarily conserved β′ Thr931for Ile. Thr931 is part of segment G, whose sequence is well conserved in largest (β′-like) RNAP subunits from bacteria to man (15Allison L.A. Moyle M. Shales M. Ingles C.J. Cell. 1985; 42: 599-610Abstract Full Text PDF PubMed Scopus (444) Google Scholar). In the structural model of RNAP core from thermophilic eubacterium Thermus aquaticus a residue equivalent toE. coli β′ Thr931 is exposed on the inner surface of RNAP secondary channel, a narrow opening that leads from RNAP surface to the catalytic center (24Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar). Based on structural considerations, the secondary channel was hypothesized to direct substrates toward the enzyme active site and to accept the 3′-end-proximal portion of the nascent RNA in transcription elongation complexes that assumed the dead-end conformation (24Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 25Korzheva N. Mustaev A. Kozlov M. Malhotra A. Nikiforov V. Goldfarb A. Darst S.A. Science. 2000; 289: 619-625Crossref PubMed Scopus (338) Google Scholar, 26Severinov K. Curr. Opin. Microbiol. 2000; 3: 118-125Crossref PubMed Scopus (46) Google Scholar). Thus, the location of the residue affected by rpoC MccJ25 resistance mutation suggests a novel mechanism of RNAP inhibition: occlusion of RNAP secondary channel. Here, we report the isolation of several MccJ25 resistance mutations in evolutionarily conserved segments G, G′, and F of cloned E. coli rpoC. The locations of the corresponding β′ residues on the T. aquaticus RNAP structure are exposed in the inside surface of RNAP secondary channel, strongly supporting the idea that MccJ25 inhibits transcription by binding to and occluding this channel. Plasmids pRW308 (27Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2927Crossref PubMed Scopus (82) Google Scholar) and pRL663 (12Severinov K. Markov D. Nikiforov V. Severinova E. Landick R. Darst S.A. Goldfarb A. J. Biol. Chem. 1995; 270: 23926-23929Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), overproducing wild-type or C-terminally hexahistidine-tagged β′ subunit, respectively, were used to obtain MccJ25-resistant mutations. MccJ25 resistant rpoC mutants generated by error-prone PCR were selected from plasmid banks described by Weilbaecher et al. (27Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2927Crossref PubMed Scopus (82) Google Scholar). To generate site-specific mutations in segment G, a derivative of pRW308 harboring a uniqueXhoI site at rpoC codon 943 was created by PCR mutagenesis. The β′ subunit encoded by the resultant plasmid, pRW308Xho_943, was wild-type because of the degeneracy of the genetic code. The rpoC positions 928, 929, 930, and 931 were next randomized using mutagenic oligonucleotides complementary torpoC codons 922–946 and incorporating a XhoI site at codon 943. At the site of randomization, positions corresponding to the first and second bases of the codon were equimolar mixtures of A, G, C, and T, whereas positions corresponding to the third base of the codon was an equimolar mixture of G and C. Mutagenic oligonucleotides were used as primers in a PCR reaction with pRW308Xho_943 template. As a second primer, an oligonucleotide whose sequence corresponded to rpoCpositions 2534–2555 was used. This primer anneals upstream of a unique pRW308 SalI site located at rpoC position 2629. After amplification, PCR fragments were treated with SalI and XhoI and ligated into appropriately treated pRW308Xho_943. Ligation mixtures were transformed in MccJ25-sensitive DH5α E. coli host cells, and transformants were plated on solid LB medium containing 200 μg/ml ampicillin. After overnight growth at 37 °C, recombinant colonies were replica-plated on LB plates containing 200 μg/ml ampicillin, 50 μg/ml MccJ25 (purified as described previously; see Ref. 20Blond A. Cheminant M. Segalas-Milazzo I. Peduzzi J. Barthelemy M. Goulard C. Salomon R. Moreno F. Farias R. Rebuffat S. Eur. J. Biochem. 2001; 268: 2124-2133Crossref PubMed Scopus (38) Google Scholar), and 1 mm IPTG to derepress the lac promoter that drives expression of plasmid-borne rpoC. MccJ25-resistant colonies were purified, and plasmid DNA was prepared and retransformed into DH5α E. coli cells. Transformants were plated on plates containing MccJ25 to confirm that resistance is plasmid-borne. An entire SalI-XhoI rpoC fragment was next sequenced at the Rockefeller University DNA technology center to establish the nature of the mutational change leading to MccJ25 resistance. To randomize rpoC codons 1136 and 1137 (evolutionarily conserved segment G′) we made use of a unique SgrAI recognition site at rpoC position 3402 (codon 1134). Mutagenic oligonucleotides spanned the SgrAI site, as well as positions to be randomized. They were used as primers with pRL663 template and another primer, whose sequence was complementary torpoC positions 3745–3777. This primer anneals downstream of a unique pRL663 BspEI site located at rpoCposition 3639. PCR fragments were treated with SgrAI andBspEI and ligated with appropriately treated pRL663, and MccJ25-resistant clones were selected and confirmed as above. In addition to the SgrAI-BspEI fragment, a portion of rpoC coding for β′ segments F and G in mutant plasmids was also sequenced, and no changes from the published sequence were observed. Construction of the β′Δ(943–1130) mutation will be described elsewhere. 2I. Artsimovitch, V. Svetlov, K. Murakami, and R. Landick, manuscript in preparation. Highly pure RNAP from MccJ25-resistant E. coli SBG231cells (18Delgado M.A. Rintoul M.R. Farias R.N. Salomon R.A. J. Bacteriol. 2001; 183: 4543-4550Crossref PubMed Scopus (139) Google Scholar) and parental MccJ25-sensitive AB259 cells were purified as described (30Polyakov A. Severinova E. Darst S.A. Cell. 1995; 8: 365-373Abstract Full Text PDF Scopus (152) Google Scholar). RNAP from Xanthomonas oryzae was purified as described in Ref. 31Nechaev S. Yuzhenkova Y. Niedziela-Majka A. Heyduk T. Severinov K. J. Mol. Biol. 2002; 320: 11-22Crossref PubMed Scopus (23) Google Scholar. RNAP fromPseudomonas aeruginosa 8882 strain (provided by Dr. A. Chakrabarty, University of Illinois College of Medicine) was purified by standard E. coli procedure without modifications.Bacillus subtilis RNAP was purified from B. subtilis PolHis cells harboring a genomic rpoCgenetically fused to hexahistidine tag (generously provided by Drs. C. P. Moran and G. Schyns, Emory University School of Medicine). RNAP was purified from cell lysates by nickel-nitrilotriacetic acid affinity chromatography followed by ion-exchange on Resource Q (Amersham Biosciences) column. Recombinant RNAP from T. aquaticus was purified from overexpressing E. colicells as described in Ref. 32Le Grice S.F. Shih C.C. Whipple F. Sonenshein A.L. Mol. Gen. Genet. 1986; 204: 229-236Crossref PubMed Scopus (26) Google Scholar. Yeast RNAP II and RNAP III were generous gifts of Dr. Sergei Borukhov (SUNY Brooklyn) and George Kassavetis (UCSD), respectively. Mutant β′ Δ(943–1130) RNAP was purified by chitin-affinity chromatography and intein-mediated removal of the chitin binding domain tag, followed by heparin affinity column chromatography, as described elsewhere.2 To partially purify RNAP containing β′ expressed from a plasmid, E. coli 397C cells (29Nedea E.C. Markov D. Naryshkina T. Severinov K. J. Bacteriol. 1999; 181: 2663-2665Crossref PubMed Google Scholar) were transformed with pRW308, pRL663, or their derivatives, grown at 30 °C in 200 ml of LB medium containing 200 μg/ml ampicillin untilA 600 of 0.5, induced with 1 mm IPTG for 4 h, collected, disrupted by sonication, and polymin P fractionation was performed as described by Kashlev et al.(28Kashlev M. Nudler E. Severinov K. Borukhov S. Komissarova N. Goldfarb A. Methods Enzymol. 1996; 274: 326-334Crossref PubMed Scopus (78) Google Scholar). 1 m NaCl extract of polymin P pellet containing ∼10% pure RNAP was precipitated with ammonium sulfate, and precipitate was stored at −80 °C. Before use, an aliquot of ammonium sulfate pellet was dissolved in transcription buffer (40 mm Tris-HCl, pH 7.9, 40 mm KCl, 10 mm MgCl2, 5% glycerol) to give a final protein concentration of ∼1 mg/ml, and this preparation was used in transcription assays. Transcription from the T7 A1 promoter-containing DNA fragment was performed in 10-μl transcription buffer reactions containing 50 ng of DNA, 0.5 μg of wild-type or mutant RNAP, 0.5 mm CpA primer, 2.5 μm α-[32P]UTP (300 Ci/mmol), and different concentrations of MccJ25. Reactions proceeded for 10 min at 37 °C and were terminated by the addition of urea-containing loading buffer. Products were analyzed by urea-PAGE electrophoresis (7m urea, 20% polyacrylamide), followed by autoradiography and PhosphorImager analysis. Transcription from B. subtilis vegA promoter (32Le Grice S.F. Shih C.C. Whipple F. Sonenshein A.L. Mol. Gen. Genet. 1986; 204: 229-236Crossref PubMed Scopus (26) Google Scholar) was performed in a buffer containing 40 mm Tris-HCl, pH 7.9, 10 mmMgCl2, 0.1 mm EDTA, 0.1 mmdithiothreitol, 5% glycerol, 25 μg/ml bovine serum albumin using 0.5 mm UpA primer and 2.5 μmα-[32P]GTP (300 Ci/mmol) substrate. Earlier, one of our groups (18Delgado M.A. Rintoul M.R. Farias R.N. Salomon R.A. J. Bacteriol. 2001; 183: 4543-4550Crossref PubMed Scopus (139) Google Scholar) reported thatE. coli cells harboring the sjmA1 mutation, but not the wild-type E. coli, were able to grow on selective medium containing MccJ25. The sjmA1 mutation was found to correspond to a substitution of Thr931 to Ile in the largest subunit of E. coli RNAP, the β′ subunit. The original report also established that MccJ25 partially inhibited a steady-state in vitro transcription by the wild-typeE. coli RNAP, strongly implying that RNAP is a direct target of MccJ25. However, RNAP harboring the T931I substitution was not tested in these experiments. The experiment presented in Fig. 1 demonstrates that the mutant enzyme is indeed resistant to MccJ25 in vitro. As can be seen, MccJ25 inhibited T7 A1 promoter-directed synthesis of the CpApU abortive RNA product from the CpA dinucleotide primer and radioactively labeled UTP by the wild-type RNAP (compare lanes 4 and 5). In contrast, the CpApU synthesis by RNAP purified from cells harboring thesjmA1 mutation was unaffected by the drug (comparelanes 1 and 2). Order-of-addition experiments established that MccJ25 inhibited abortive RNA synthesis when added either before or after the formation of open promoter complex on the T7 A1 promoter-containing DNA fragment used as a template in this experiment (compare lanes 5 and 6). We therefore conclude that (i) RNAP is a true cellular target of MccJ25, and (ii) MccJ25 does not act by preventing RNAP interaction with DNA. The genetic context of thesjmA1 mutation is shown in Fig. 3. As can be seen, the corresponding substitution occurred in a highly conserved segment of the E. coli β′ subunit, segment G. We hypothesize that the T931I substitution causes MccJ25 resistance by preventing MccJ25 binding to RNAP and that Thr931 is a part of MccJ25 binding site. Given the very high level of evolutionary conservation of segment G, the following two questions are of interest. First, can other MccJ25-resistant mutations in segment G be obtained? Second, will MccJ25 inhibit RNAPs from organisms other than E. coli? To answer the first question, we obtained plasmids expressing mutantrpoC genes, transformed these plasmids into MccJ25-sensitiveE. coli cells, and checked the ability of plasmid-bearing cells to grow on a medium containing MccJ25. In case when growth on selective medium was observed, we purified RNAPs containing mutant β′ and confirmed that mutant RNAPs were indeed resistant to MccJ25. In cases when no in vivo resistance was observed, we considered the possibility that RNAP containing mutant β′ could not support cell growth in the presence of MccJ25, when the wild-type, chromosomally encoded RNAP was inactivated. Therefore, RNAPs containing plasmid-borne β′ were also purified, and their sensitivity to MccJ25 was testedin vitro. All mutants reported below were tested this way. Fig. 2 shows the results of in vivo and complementary in vitro testing with some of the mutants as an example. A set of several point mutations in segment G of E. coli rpoC cloned on an expression plasmid was recovered in two unrelated screens, one aimed at obtaining termination-alteringrpoC mutants (27Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2927Crossref PubMed Scopus (82) Google Scholar) and another site-specifically mutating evolutionarily conserved β′ positions 921 and 935,2 is presented atop of the sequence alignment shown in Fig. 3. MccJ25-sensitiveE. coli cells were transformed with plasmids expressing mutant rpoC genes, and the ability of plasmid-bearing cells to grow on MccJ25-containing medium was investigated. As controls, cells harboring plasmids expressing wild-type rpoC or MccJ25-resistant rpoCT931I allele were employed. As expected, cells expressing wild-type rpoC were sensitive to MccJ25, whereas cells expressing the T931I allele were resistant (Fig. 2 and data not shown). Cells harboring expression plasmids bearing the F935S allele were as resistant as control cells expressing rpoC T931I, whereas cells expressing the R933H,A946V double mutant resulted in slow but detectable growth on MccJ25-containing medium (Fig. 2 and data not shown). In contrast, cells expressingQ921P, T934M, and H936Y alleles did not grow on selective medium (Fig. 2 and data not shown). The results of in vitro transcription assays correlated with the in vivo results (Fig. 2) (data not shown). However, theR933H,A946V double mutant, which showed low levels of resistance in vivo, was highly resistant in vitro, suggesting that the mutant RNAP in vivo function is impaired. RNAP harboring the F935S substitution was found to be resistant to the drug, whereas other mutants were sensitive. Three RNAP harboring dominant lethal mutations in segment G, M932L, R933S, and T934A, were also tested for MccJ25 resistance. These mutants were obtained in the course of an independent mutagenesis effort 3V. Epshtein, A. Mustaev, and A. Goldfarb, submitted for publication. and were prepared byin vitro reconstitution. Additional MccJ25-resistant mutants in segment G were also sought directly. Three rpoC codons immediately to the left of position 931 (928, 929, and 930) were randomized by site-directed PCR mutagenesis, libraries of recombinant plasmids were transformed in MccJ25-sensitive E. coli cells, and MccJ25-resistant clones were selected. As a control, position 931, the site of the original MccJ25-resistant mutation, was also randomized. MccJ25-resistant clones were only obtained in the control mutagenesis reaction. Sequencing of three resistant clones revealed the presence of the original mutation, T931I, as well as two new mutations, T931N and T931L. The corresponding enzymes were also resistant in vitro (data not shown). The result thus suggests that the identity of β′ amino acids 928–930 is either not important for MccJ25 inhibition, or MccJ25-resistant substitutions at these positions lead to lethal phenotype. MccJ25 is effective against Gram-negative bacteria but has no effect on Gram-positive bacteria (19Salomon R.A. Farias R.N. J. Bacteriol. 1992; 174: 7428-7435Crossref PubMed Google Scholar). To determine the specificity of transcription inhibition by MccJ25, we assembled a panel of RNAPs prepared from several Gram-negative and Gram-positive bacteria and compared their ability to perform abortive RNA synthesis in the presence or in the absence of MccJ25 (Fig. 4). In the absence of MccJ25, RNAPs from Gram-negative bacteria demonstrated approximately equal specific activities on the T7 A1 promoter (0.9, 0.6, 1.2, and 0.8 pmol/min of CpApU synthesized by 1 pmol of wild-type E. coliRNAP, E. coli RNAPT931I, P. aeruginosa RNAP, and X. oryzae RNAP, respectively). In agreement with the previously determined in vivospecificity, MccJ25 inhibited abortive synthesis of CpApU from the T7 A1 promoter-containing DNA fragment by RNAPs prepared from three Gram-negative bacteria, wild-type E. coli, X. oryzae, and P. aeruginosa (see Fig. 4; 10, 11, and 9% residual activity in the presence of 25 μm MccJ25, respectively). As expected, E. coli RNAPT931Iwas active in the presence of 25 μm MccJ25 (85% activity). RNAP from T. aquaticus was assayed on the T7 A1 promoter at 60 °C and was considerably less active (0.2 pmol of CpApU synthesized per min per pmol of enzyme). MccJ25 had no effect on abortive synthesis by recombinant T. aquaticus RNAP at 60 °C (see Fig. 4; 100% activity in the presence of 25 μm MccJ25). Because RNAP from B. subtilis displays only a very low level of activity on the T7 A1 promoter (data not shown), we assayed the effect of MccJ25 on this enzyme during the abortive synthesis of UpApG on B. subtilis vegA promoter (32Le Grice S.F. Shih C.C. Whipple F. Sonenshein A.L. Mol. Gen. Genet. 1986; 204: 229-236Crossref PubMed Scopus (26) Google Scholar). In the absence of MccJ25,B. subtilis RNAP, E. coli RNAP, and E. coli RNAPT931I demonstrated comparable levels of activity on the vegA promoter (0.8, 0.3, and 0.1 pmol of UpApG synthesized per min per pmol of RNAP, respectively). MccJ25 had no effect on the B. subtilis enzyme (see Fig. 4; 110% activity in the presence of 25 μm MccJ25) and E. coli RNAPT931I (see Fig. 4; 85% activity in the presence of 25 μm MccJ25) but was active against wild-type E. coli enzyme on this promoter (see Fig. 4; 11% activity in the presence of 25 μm MccJ25). Additional experiments demonstrated that MccJ25 had no effect on transcription by yeast RNAPs II and III (data not shown). Because segment G positions affected by MccJ25-resistant substitutions are identical in β′ homologues from Gram-positive and Gram-negative organisms, the result implies that other regions of RNAP may also contribute to MccJ25 binding. In RNAP from Gram-negative bacteria, segment G is followed by a long stretch of amino acid sequence that is hypervariable in evolution (33Zakharova N. Bass A. Arsenieva E. Nikiforov V. Severinov K. J. Biol. Chem. 1998; 273: 24912-24920Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The hypervariable region is missing in RNAPs from Gram-positive bacteria and eukaryal RNAPs. To test whether the presence of the evolutionarily hypervariable region of β′ contributes to the MccJ25 sensitivity of RNAP from Gram-negative bacteria, we tested the ability ofrpoCΔ(943–1130) allele that lacks the entire hypervariable region and thus resembles homologues from Gram-positive microorganisms to confer MccJ25 resistance in vivo. The mutant β′ poorly assembles into RNAP, presumably because of its inability to compete with chromosomally encoded wild-type β′. 4I. Artsimovitch, personal observation. We therefore tested the ability of MccJ25-sensitive cells harboring plasmid pIA331, which, in the presence of IPTG, co-overexpresses wild-type rpoA(α), rpoB (β), andrpoCΔ(943–1130) and thus increases the efficiency of the mutant enzyme assembly to grow on MccJ25-containing medium. As controls, plasmid pIA423, which co-overexpresses wild-typerpoA, rpoB, and rpoC, and pRL663rpoC + and pRL663rpoCT931Iplasmids, were used. As can be seen from Fig. 5 A, cells harboring pIA331 and pRL663T931I, but not cells harboring pIA423 and pRL663, formed colonies in the presence of MccJ25 and IPTG. Colonies formed by cells harboring pIA331 were minute as compared with colonies formed by cells harboring pRL663rpoCT931I cells, but the efficiency of plating was comparable. Plasmid pIA331, but not other plasmids, significantly inhibited cell growth in the presence of IPTG only, suggesting that RNAPΔ(943–1130) was defective in some cellular function(s) unrelated to MccJ25 resistance. Be that as it may, the results demonstrate that hypervariable region indeed contributes to MccJ25 sensitivity and may be partially dispensable for cell viability at our conditions, because RNAP lacking β′ residues 943–1130 is presumably the only transcriptionally active enzyme in the presence of MccJ25. E. coli RNAPΔ(943–1130) was prepared from cells harboring pIA331 and tested for the ability to transcribe from the T7 A1 promoter in the presence or in the absence of MccJ25 (Fig. 5 B). The results showed that the mutant was more resistant to the drug than wild-type E. coli RNAP (26 and 3% residual activity in the presence of 50 μm MccJ25). Because RNAPs from Gram-positive bacteria are highly resistant to MccJ25 in vitro, other sites in these RNAPs must contribute to MccJ25 resistance. In the β′ subunits from Gram-negative bacteria, the hypervariable segment is followed by evolutionarily conserved segment G′ (33Zakharova N. Bass A. Arsenieva E. Nikiforov V. Severinov K. J. Biol. Chem. 1998; 273: 24912-24920Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Because there is no hypervariable region in homologues from Gram-positive bacteria, segments G and G′ form a continuous stretch of evolutionary conserved sequence in the β′ subunits from these organisms (Fig. 3). Segment G residues that cause MccJ25 resistance are part of the so-called G-loop in RNAP structures from thermophilic bacteria of the Thermus genus (24Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 34Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. Borukhov S. Yokoyama S. Nature. 2002; 417: 712-719Crossref PubMed Scopus (631) Google Scholar, 35Murakami K.S. Masuda S. Darst S.A. Science. 2002; 296: 1280-1284Crossref PubMed Scopus (449) Google Scholar). Residues of segment G′ are also part of the G-loop. In particular,Thermus RNAP residues corresponding to E. coliβ′ amino acids 1137 and 1138 are in direct contact with the residue corresponding to E. coli Thr931 and are located at the base of the G-loop. We therefore considered a possibility that substitutions in positions 1137 and 1138 will make E. coliRNAP MccJ25-resistant. Accordingly, codons 1137 and 1138 of plasmid-borne rpoC were randomized, mutant plasmid libraries were transformed in MccJ25-sensitive E. coli, and transformants were plated on selective medium containing MccJ25. No MccJ25-resistant mutants were obtained when codon 1137 was randomized. One clone was picked up at random and found to encode a G1137A substitution; the corresponding RNAP was MccJ25-sensitive in vitro. One resistant clone, coding for L1138T, was recovered from codon 1138 mutagenesis. The corresponding RNAP was purified and found to be MccJ25-resistant in vitro (Fig. 2). Because no changes from the published rpoC sequence in segment G was observed in this mutant (data not shown), we conclude that a substitution in segment G′ is indeed responsible for MccJ25 resistance. One MccJ25-sensitive clone from the 1138 mutagenesis reaction was picked up at random and sequenced and found to contain a mutation coding for L1138V substitution. The corresponding RNAP was purified and found to be MccJ25-sensitive in vitro (data not shown). A functional deletion of β′ amino acids 1145–1198 immediately to the right of segment G′ was described by us previously (33Zakharova N. Bass A. Arsenieva E. Nikiforov V. Severinov K. J. Biol. Chem. 1998; 273: 24912-24920Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This deletion did not result in MccJ25 resistance in vivo and in vitro (data not shown). A double mutation coding for E1030K and I1134D substitutions was isolated in an independent PCR-based screen for termination-alteringrpoC mutations (27Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2927Crossref PubMed Scopus (82) Google Scholar). The first substitution, of Glu1030, occurred in the hypervariable region; the second substitution, of Ile1134, occurred in segment G′. We tested the ability of plasmid-borne E1030K,I1134D allele to confer MccJ25 resistance in vivo and in vitroand observed no resistance (data not shown). We also looked for additional MccJ25-resistant mutations within the bank ofrpoC expression plasmids subjected to error-prone PCR at and around segments G and G′ (rpoC codons 876–1213; see Ref.27Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2927Crossref PubMed Scopus (82) Google Scholar). A triple mutation coding for I1115V, G1136D, and F1145S substitutions was recovered in this way. Of the three residues affected, one (β′ Phe1145) is removed by MccJ25-sensitive Δ(1145–1198) deletion. Substitutions I1115V and/or G1136D are thus likely responsible for MccJ25 resistance. Because I1115V is a conservative substitution, substitution of evolutionarily conserved Gly1136 in segment G′ is the probable cause of MccJ25 resistance. Residues of β′ segments G and G′ that are important for MccJ25 inhibition are exposed on the surface of narrow RNAP secondary channel that opens on the downstream face of the enzyme and leads to the catalytic site (24Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar). In addition to β′ segments G and G′, conserved segment F also participates in the formation of the secondary channel. We were therefore interested in whether MccJ25-resistant mutations in segment F can be obtained. Toward this end, we tested two segment F mutants, F773I and S793F, that were shown previously to cause resistance to the elongation inhibitor, streptolydigin (12Severinov K. Markov D. Nikiforov V. Severinova E. Landick R. Darst S.A. Goldfarb A. J. Biol. Chem. 1995; 270: 23926-23929Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These mutants did not result in appreciable MccJ25 resistance in vivo or in vitro (Fig. 2) (data not shown). We therefore looked for MccJ25-resistant region F mutants directly, by incorporating an error-prone PCR-amplified rpoCfragment coding for region F (rpoC codons 544–875) into anrpoC expression plasmid, transforming mutant plasmids in MccJ25-sensitive host, and selecting MccJ25-resistant colonies. Several independent MccJ25-resistant colonies were obtained, and the plasmid-borne nature of MccJ25 resistance was confirmed by retransforming of rpoC expression plasmids from MccJ25-resistant clones into sensitive host and replating on selective medium. Four independent clones were obtained, and their sequence at and around segments F, G, and G′ was determined. No changes in segment G/G′ sequences was detected. In contrast, changes from the published sequence leading to substitutions of segment F residues Ser733 for Pro, Leu783 for Gln, and a double substitution of Leu746 for Pro and Phe773 for Ile were observed (Fig. 6). In vitro analysis confirmed that RNAPs carrying mutations in segment F are resistant to MccJ25 (Fig. 2) (data not shown). We conclude that substitutions in RNAP β′ segment F lead to MccJ25 resistance. Substitutions in β′ segment F that lead to MccJ25 resistance occurred close to segment F sites that, when mutated, cause Stl resistance (12Severinov K. Markov D. Nikiforov V. Severinova E. Landick R. Darst S.A. Goldfarb A. J. Biol. Chem. 1995; 270: 23926-23929Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The main cluster of Stl-resistant mutations is located in the β subunit, between Rif clusters I and II (3Severinov K. Soushko M. Goldfarb A. Nikiforov V. J. Biol. Chem. 1993; 268: 14280-14825Abstract Full Text PDF Google Scholar). To further investigate the relationship between MccJ25 and Stl resistance we tested the ability of E. coli cells expressing several MccJ25-resistant alleles to grow in the presence of Stl, and we tested the ability of cells expressing Stl-resistant rpoB(β) alleles to grow in the presence of MccJ25. As expected, cells overproducing Stl-resistant β′ harboring S793F substitution, as well as cells overproducing Stl-resistant β subunit microdeletionsΔ(540–544) andΔ(540L545) (3Severinov K. Soushko M. Goldfarb A. Nikiforov V. J. Biol. Chem. 1993; 268: 14280-14825Abstract Full Text PDF Google Scholar), but not cells overproducing wild-type β′ or β, grew on plates containing Stl. Also as expected, cells overproducing partially Stl-resistant βΔ(535–542) formed minute colonies in the presence of Stl, whereas cells overproducing Stl-sensitive βΔ(538–540) did not grow (3Severinov K. Soushko M. Goldfarb A. Nikiforov V. J. Biol. Chem. 1993; 268: 14280-14825Abstract Full Text PDF Google Scholar) (data not shown). None of the segment F, segment G, or segment G′ MccJ25-resistantrpoC mutations tested allowed growth on Stl-containing plates (data not shown). Plating of cells expressing Stl-resistant rpoB alleles on MccJ25 gave an unexpected result. As expected cells expressingrpoCT931I, but not cells expressing wild-typerpoC or Stl-resistant rpoCS793F, grew in the presence of MccJ25 (Fig. 7). Likewise, cells expressing wild-type rpoB and Stl-sensitiverpoBΔ(538–540) did not grow in the presence of MccJ25. In contrast, cells expressing highly Stl-resistantΔ(540–544) andΔ(540L545) grew in the presence of MccJ25, whereas cells expressing low-level Stl resistance allelerpoBΔ(535–542) formed minute colonies. The results of the plating assay were supported by the results of in vitro transcription experiments (data not shown). We also tested several plasmid-borne Rifampicin-resistant rpoB mutants and found that none of them were able to support growth in the presence of MccJ25 (data not shown). We conclude that Stl-resistant mutations in the rpoB gene lead to MccJ25 resistance. The principal result of this work is the demonstration that transcription by mutant RNAP purified from MccJ25-resistant E. coli cells is resistant to MccJ25, whereas transcription by RNAP purified from wild-type cells is MccJ25-sensitive. This result proves that E. coli RNAP is the cellular target of MccJ25. In the best understood case of Rifampicin, mutations toward resistance affect RNAP residues that are removed from each other in primary sequence but that cluster in the enzyme quaternary structure (1Ovchinnikov Y.A. Monastyrskaya G.S. Guriev S.O. Kalinina N.F. Sverdlov E.D. Gragerov A.I. Bass I.A. Kiver I.F. Moiseyeva E.P. Igumnov V.N. Mindlin S.Z. Nikiforov V.G. Khesin R.B. Mol. Gen. Genet. 1983; 190: 344-348Crossref PubMed Scopus (80) Google Scholar, 2Jin D.J. Gross C.A. J. Mol. Biol. 1988; 202: 45-58Crossref PubMed Scopus (545) Google Scholar, 3Severinov K. Soushko M. Goldfarb A. Nikiforov V. J. Biol. Chem. 1993; 268: 14280-14825Abstract Full Text PDF Google Scholar, 24Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar). Structural analysis demonstrates that Rif-resistance mutation define the Rifampicin binding site (4Campbell E.A. Korzheva N. Mustaev A. Murakami K. Nair S. Goldfarb A. Darst S.A. Cell. 2001; 104: 901-912Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar). We hypothesized that the original MccJ25-resistance mutation, that changed evolutionarily conserved amino acid inside RNAP secondary channel, likewise defines the MccJ25 binding site on RNAP. According to this view, MccJ25 inhibits transcription by binding in the secondary channel and preventing the traffic of NTP substrates to the catalytic center of the enzyme. Indeed, molecular modeling using T. aquaticus RNAP structure and a reported MccJ25 structure (20Blond A. Cheminant M. Segalas-Milazzo I. Peduzzi J. Barthelemy M. Goulard C. Salomon R. Moreno F. Farias R. Rebuffat S. Eur. J. Biochem. 2001; 268: 2124-2133Crossref PubMed Scopus (38) Google Scholar) shows that 21-amino acid MccJ25 can fit into RNAP secondary channel with little or no steric clashes (data not shown).