Nucleotides from –16 to –12 Determine Specific Promoter Recognition by Bacterial σS-RNA Polymerase
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m305281200
ISSN1083-351X
AutoresStéphan Lacour, Annie Kolb, Paolo Landini,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe alternative sigma factor σS, mainly active in stationary phase of growth, recognizes in vitro a –10 promoter sequence almost identical to the one for the main sigma factor, σ70, thus raising the problem of how specific promoter recognition by σS-RNA polymerase (EσS) is achieved in vivo. We investigated the promoter features involved in selective recognition by EσS at the strictly σS-dependent aidB promoter. We show that the presence of a C nucleotide as first residue of the aidB –10 sequence (–12C), instead of the T nucleotide canonical for σ70-dependent promoters, is the major determinant for selective recognition by EσS. The presence of the –12C does not allow formation of an open complex fully proficient in transcription initiation by Eσ70. The role of –12C as specific determinant for promoter recognition by EσS was confirmed by sequence analysis of known EσS-dependent promoters as well as site-directed mutagenesis at the promoters of the csgB and sprE genes. We propose that EσS, unlike Eσ70, can recognize both C and T as the first nucleotide in the –10 sequence. Additional promoter features such as the presence of a C nucleotide at position –13, contributing to open complex formation by EσS, and a TG motif found at the unusual –16/–15 location, possibly contributing to initial binding to the promoter, also represent important factors for σS-dependent transcription. We propose a new sequence, TG(N)0–2CCATA(c/a)T, as consensus –10 sequence for promoters exclusively recognized by EσS. The alternative sigma factor σS, mainly active in stationary phase of growth, recognizes in vitro a –10 promoter sequence almost identical to the one for the main sigma factor, σ70, thus raising the problem of how specific promoter recognition by σS-RNA polymerase (EσS) is achieved in vivo. We investigated the promoter features involved in selective recognition by EσS at the strictly σS-dependent aidB promoter. We show that the presence of a C nucleotide as first residue of the aidB –10 sequence (–12C), instead of the T nucleotide canonical for σ70-dependent promoters, is the major determinant for selective recognition by EσS. The presence of the –12C does not allow formation of an open complex fully proficient in transcription initiation by Eσ70. The role of –12C as specific determinant for promoter recognition by EσS was confirmed by sequence analysis of known EσS-dependent promoters as well as site-directed mutagenesis at the promoters of the csgB and sprE genes. We propose that EσS, unlike Eσ70, can recognize both C and T as the first nucleotide in the –10 sequence. Additional promoter features such as the presence of a C nucleotide at position –13, contributing to open complex formation by EσS, and a TG motif found at the unusual –16/–15 location, possibly contributing to initial binding to the promoter, also represent important factors for σS-dependent transcription. We propose a new sequence, TG(N)0–2CCATA(c/a)T, as consensus –10 sequence for promoters exclusively recognized by EσS. Bacterial cells adapt to changing environmental and physiological conditions by modulating gene expression. Sigma (σ) factors of RNA polymerase, as the subunits responsible for promoter recognition, play a major role in programming gene expression. At least seven different σ subunits have been identified in Escherichia coli; σ70 is the main σ subunit and can carry out transcription from the majority of E. coli promoters. The alternative σ subunits can direct transcription toward specific sets of genes (i.e. heat-shock, extracellular proteins, etc.) whose transcription is directed by σ-specific promoter sequences. A partial exception to the typical role for alternative σ subunits is represented by σS, the product of the rpoS gene, mainly expressed in the stationary phase of growth (1Lange R. Hengge-Aronis R. Genes Dev. 1994; 8: 1600-1612Crossref PubMed Scopus (415) Google Scholar, 2Jishage M. Ishihama A. J. Bacteriol. 1995; 177: 6832-6835Crossref PubMed Scopus (185) Google Scholar, 3Jishage M. Iwata A. Ueda S. Ishihama A. J. Bacteriol. 1996; 178: 5447-5451Crossref PubMed Google Scholar). Unlike the other σ subunits, σS-RNA polymerase (EσS) 1The abbreviations used are: EσS, σS-RNA polymerase; Eσ70, σ70-RNA polymerase; PaidB, aidB promoter. can initiate transcription from several promoters also recognized by Eσ70, suggesting that they recognize similar promoter sequences (4Tanaka K. Takayanagi Y. Fujita N. Ishihama A. Takahashi H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3511-3515Crossref PubMed Scopus (193) Google Scholar). The recognition of similar promoter sequences by σS and σ70 is reflected by their strong similarity in the DNA binding domains (5Lonetto M. Gribskov M. Gross C.A. J. Bacteriol. 1992; 174: 3843-3849Crossref PubMed Scopus (737) Google Scholar). The alignment of EσS-dependent promoters and the search for an optimal promoter for EσS in vitro using the systematic evolution of ligands by exponential enrichment (SELEX) procedure have pointed to a –10 consensus sequence for EσS, CTATA(c/a)T that is very similar to the canonical TATAAT sequence for σ70 (6Hawley D.K. McClure W.R. Nucleic Acids Res. 1983; 11: 2237-2255Crossref PubMed Scopus (1488) Google Scholar, 7Espinosa-Urgel M. Chamizo C. Tormo A. Mol. Microbiol. 1996; 21: 657-659Crossref PubMed Scopus (103) Google Scholar, 8Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Crossref PubMed Google Scholar, 9Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Gaal T. Ross W. Estrem S.T. Nguyen L.H. Burgess R.R. Gourse R.L. Mol. Microbiol. 2001; 42: 939-954Crossref PubMed Scopus (150) Google Scholar). These results suggest that promoter selectivity between σ70 and σS might be determined by factors other than promoter DNA sequence. Indeed, studies of different σS-dependent promoters suggest that other parameters such as increased intracellular salt concentration, degree of DNA supercoiling, the ppGpp alarmone, and modulation by trans-acting regulators all contribute to σS selectivity (reviewed in Refs. 11Hengge-Aronis R. Curr. Opin. Microbiol. 1999; 2: 148-152Crossref PubMed Scopus (188) Google Scholar, 12Hengge-Aronis R. J. Mol. Microbiol. Biotechnol. 2002; 4: 341-346PubMed Google Scholar, 13Hengge-Aronis R. Curr. Opin. Microbiol. 2002; 5: 591-595Crossref PubMed Scopus (115) Google Scholar, 14Ishihama A. Annu. Rev. Microbiol. 2000; 54: 499-518Crossref PubMed Scopus (454) Google Scholar, 15Vicente M. Chater K.F. De Lorenzo V. Mol. Microbiol. 1999; 33: 8-17Crossref PubMed Scopus (52) Google Scholar). The main sequence feature specific for σS-dependent promoters would be a C nucleotide immediately upstream of the –10 promoter element (CTATA(c/a)T at the –13 position relative to the transcription start (–13C)); the –35 element does not appear to be important for promoter recognition by σS, although conflicting data on its role have been reported (10Gaal T. Ross W. Estrem S.T. Nguyen L.H. Burgess R.R. Gourse R.L. Mol. Microbiol. 2001; 42: 939-954Crossref PubMed Scopus (150) Google Scholar, 16Tanaka K. Kusano S. Fujita N. Ishihama A. Takahashi H. Nucleic Acids Res. 1995; 23: 827-834Crossref PubMed Scopus (104) Google Scholar, 17Hiratsu K. Shinagawa H. Makino K. Mol. Microbiol. 1995; 18: 841-850Crossref PubMed Scopus (39) Google Scholar, 18Wise A. Brems R. Ramakrishnan V. Villarejo M. J. Bacteriol. 1996; 178: 2785-2793Crossref PubMed Google Scholar, 19Colland F. Fujita N. Kotlarz D. Bown J.A. Meares C.F. Ishihama A. Kolb A. EMBO J. 1999; 18: 4049-4059Crossref PubMed Scopus (51) Google Scholar). Mutagenesis studies on σS-dependent promoters have shown that deviations from the consensus CTATACT affect promoter recognition by σS in vivo and have substantiated the importance of the –13C element (8Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Crossref PubMed Google Scholar, 20Bordes P. Repoila F. Kolb A. Gutierrez C. Mol. Microbiol. 2000; 35: 845-853Crossref PubMed Scopus (49) Google Scholar, 21Lacour S. Kolb A. Zehnder A.J.B. Landini P. Biochem. Biophys. Res. Commun. 2002; 292: 922-930Crossref PubMed Scopus (12) Google Scholar). Suppression genetics data suggest that the –13C is directly contacted by the residue Lys-173 of region 2.5 of σS (8Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Crossref PubMed Google Scholar). Interestingly, the amino acid located at the equivalent position in region 2.5 of σ70 is a glutamate residue (Glu-458) that has been proposed to interact with another promoter element located upstream of the –10, the TG motif (22Barne K.A. Bown J.A. Busby S.J. Minchin S.D. EMBO J. 1997; 16: 4034-4040Crossref PubMed Scopus (232) Google Scholar). At the so-called “extended –10” promoters, this TG dinucleotide can be found two nucleotides upstream from the –10 sequence, where it allows transcription even in the absence of a conserved –35 sequence (23Ponnambalam S. Webster C. Bingham A. Busby S. J. Biol. Chem. 1986; 261: 16043-16048Abstract Full Text PDF PubMed Google Scholar, 24Keilty S. Rosenberg M. J. Biol. Chem. 1987; 262: 6389-6395Abstract Full Text PDF PubMed Google Scholar, 25Minchin S. Busby S. Biochem. J. 1993; 289: 771-775Crossref PubMed Scopus (23) Google Scholar, 26Kumar A. Malloch R.A. Fujita N. Smillie D.A. Ishihama A. Hayward R.S. J. Mol. Biol. 1993; 232: 406-418Crossref PubMed Scopus (210) Google Scholar, 27Bown J.A. Owens J.T. Meares C.F. Fujita N. Ishihama A. Busby S.J. Minchin S.D. J. Biol. Chem. 1999; 274: 2263-2270Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 28Burr T. Mitchell J. Kolb A. Minchin S. Busby S. Nucleic Acids Res. 2000; 28: 1864-1870Crossref PubMed Scopus (113) Google Scholar). However, in vitro selection experiments (10Gaal T. Ross W. Estrem S.T. Nguyen L.H. Burgess R.R. Gourse R.L. Mol. Microbiol. 2001; 42: 939-954Crossref PubMed Scopus (150) Google Scholar) suggest that a TG motif might also be recognized by σS and have proposed TGTGCTATA(c/a)T as the optimal extended –10 sequence for σS binding. The aidB gene belongs to the adaptive response to DNA alkylating agents and is activated by the Ada regulatory protein (29Volkert M.R. Nguyen D.C. Beard K.C. Genetics. 1986; 112: 11-26Crossref PubMed Google Scholar) (for reviews, see Refs. 30Landini P. Volkert M.R. J. Bacteriol. 2000; 182: 6543-6549Crossref PubMed Scopus (65) Google Scholar and 31Volkert M.R. Landini P. Curr. Opin. Microbiol. 2001; 4: 178-185Crossref PubMed Scopus (31) Google Scholar). The Ada protein, in its methylated form (meAda), can activate transcription at the aidB promoter (PaidB) by both the Eσ70 and EσS forms of RNA polymerase (32Landini P. Volkert M.R. J. Biol. Chem. 1995; 270: 8285-8289Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 33Landini P. Bown J.A. Volkert M.R. Busby S.J. J. Biol. Chem. 1998; 273: 13307-13312Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, in the absence of activation by Ada, aidB expression is strictly σS-dependent in vivo, and only the EσS form of RNA polymerase can efficiently carry out transcription from PaidB in vitro (34Volkert M.R. Hajec L.I. Matijasevic Z. Fang F.C. Prince R. J. Bacteriol. 1994; 176: 7638-7645Crossref PubMed Google Scholar, 35Landini P. Hajec L.I. Nguyen L.H. Burgess R.R. Volkert M.R. Mol. Microbiol. 1996; 20: 947-955Crossref PubMed Scopus (32) Google Scholar). Although Eσ70 can bind PaidB in vitro in the absence of any activator, binding by Eσ70 results in an unusual complex that is partially resistant to heparin challenge but unable to carry out full promoter opening and transcription initiation (21Lacour S. Kolb A. Zehnder A.J.B. Landini P. Biochem. Biophys. Res. Commun. 2002; 292: 922-930Crossref PubMed Scopus (12) Google Scholar). In this work, we investigate the importance of nucleotides around the –10 region of the aidB promoter for σS selectivity. We confirm the role of –13C for σS activity and show that a TG motif at the unusual –15/–16 position plays a specific role in σS-dependent transcription. We show that, unlike σ70, σS can recognize a C residue instead of the canonical T as first nucleotide of the –10 box (–12C). These features are not unique to the aidB promoter, but they are conserved in a subset of σS-dependent promoters. Thus, based on the presence of either a C oraTasthe first nucleotide in the –10 box, we can distinguish between specific EσS-dependent promoters and promoters that can be recognized by both EσS and Eσ70, at which additional regulatory factors are likely to play a role in selective promoter recognition by EσS. We propose TG(N)CCATA(c/a)T as the new consensus sequence for the factor-independent class of σS-dependent promoters. Strains and Plasmids—The strains used in this study are the E. coli K12 strains MV1161 and its rpoS derivative MV2792 (34Volkert M.R. Hajec L.I. Matijasevic Z. Fang F.C. Prince R. J. Bacteriol. 1994; 176: 7638-7645Crossref PubMed Google Scholar). These strains were transformed with derivatives of the reporter plasmid pRS1274 (36Landini P. Hajec L.I. Volkert M.R. J. Bacteriol. 1994; 176: 6583-6589Crossref PubMed Google Scholar) carrying lacZ fusions under the control of either the wild type aidB promoter or its mutant derivatives. To produce the reporter plasmids, we cloned the aidB promoter region into the multiple cloning site of the pRS1274 vector as 238-bp BamHI-EcoRI fragments. The PaidB wild type fragment was generated by PCR using the aidBbam primer (5′-TATAGCAAGCTTCGTGCGGAATGGGGATCC-3′), annealing at –140 to –123 of PaidB, and aidBeco (introducing an EcoRI site, 5′-CGGAAAGAATTCGCAGAGCGCGCCATCAGA-3′), annealing at positions 93–116. The aidB mutant promoters shown in Fig. 1 were generated by PCR using the primers aidBeco together with the appropriated mutagenic primer MaidBnco (5′-AATCCATGGCAGTgaccATACTaATGG-3′; the small italic letters indicate the position of the various substitutions), annealing at –29 to –2 and encompassing the NcoI restriction site of PaidB. The PCR products were cloned into the plasmid pMV120 (pUC18, carrying the aidB promoter region; Ref. 36Landini P. Hajec L.I. Volkert M.R. J. Bacteriol. 1994; 176: 6583-6589Crossref PubMed Google Scholar) using the NcoI and EcoRI restriction sites, and introduction of the desired mutations was checked by sequencing. Finally, the 238-bp BamHI-EcoRI fragments encompassing either the wild type or the mutant aidB promoters were subcloned into either the pRS1274 plasmid (for in vivo transcription assays) or the pJCD01 plasmid (37Marschall C. Labrousse V. Kreimer M. Weichart D. Kolb A. Hengge-Aronis R. J. Mol. Biol. 1998; 276: 339-353Crossref PubMed Scopus (92) Google Scholar) for in vitro transcription experiments (see below). In Vivo Transcription Assays—Bacterial cultures grown overnight in Luria broth (LB) medium at 37 °C were diluted 1:100 in LB, grown to an OD600 nm = 0.2, then re-diluted 1:100 in pre-warmed LB. The second dilution was performed to reduce the β-galactosidase activity carried over from the overnight cultures. Samples were collected when the re-diluted cultures reached OD600 nm = 0.1 and then after4hof growth; at this time, OD600 nm was ∼2.5 for both MV1161 and MV2792 strains. β-galactosidase activity from the aidB::lacZ fusions was determined as described previously (38Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) and was comparable with the activity obtained by a promoterless lacZ gene in samples taken at OD600 nm = 0.1 (data not shown). Protein Purification and Holoenzyme Reconstitution—Core enzyme and σ factors were purified as described previously (Refs. 39Gribskov M. Burgess R.R. Gene. 1983; 26: 109-118Crossref PubMed Scopus (130) Google Scholar and 16Tanaka K. Kusano S. Fujita N. Ishihama A. Takahashi H. Nucleic Acids Res. 1995; 23: 827-834Crossref PubMed Scopus (104) Google Scholar, respectively); proteins appeared to be pure from contaminants as determined from denaturing protein gel electrophoresis. Reconstitution of active holoenzymes for the different experiments was achieved by incubating the core enzyme and either σS or σ70 at a 1:4 ratio (to ensure saturation of the core enzyme by σ factors) for 30 min at 37 °C. For the competition experiment in a bandshift assay, the ratio σ S:σ70:core was reduced to 1, and reconstitution time was reduced to 10 min. The reconstituted holoenzymes were diluted at room temperature in K-glu200 buffer (40 mm HEPES, pH 8.0, 10 mm magnesium chloride, 200 mm potassium glutamate, 4 mm dithiothreitol, and 500 μg/ml bovine serum albumin) prior to their use for in vitro experiments. Dithiothreitol was omitted from the buffer for permanganate (KMnO4) reactivity experiments. In Vitro Transcription Assays—Single-round transcription assays were performed in K-glu200 buffer on derivatives of the supercoiled plasmid pJCDO1 (37Marschall C. Labrousse V. Kreimer M. Weichart D. Kolb A. Hengge-Aronis R. J. Mol. Biol. 1998; 276: 339-353Crossref PubMed Scopus (92) Google Scholar) carrying either wild type or mutant PaidB. Plasmids (3 nm) and reconstituted RNA polymerase holoenzymes (50 nm for EσS or 100 nm for Eσ70) were incubated for 15 min at 37 °C to allow complex formation. Elongation was started by the addition of a prewarmed mixture containing nucleotides and heparin (final concentrations were 500 μm ATP, GTP, and CTP; 30 μm UTP; 0.5 μCi of [α-32P]UTP; and 500 μg/ml heparin) to the template-polymerase mix and allowed to proceed for 10 min at 37 °C. Reactions were stopped by the addition of 10 μl of loading buffer (formamide containing 20 mm EDTA, xylene cyanol, and bromphenol blue). After heating to 65 °C, samples were loaded on 7% polyacrylamide sequencing gels. Reaction products from PaidB were quantified using a PhosphorImager (Molecular Dynamics) and normalized to the standard RNAI product after background subtraction. Other in Vitro Experiments—Linear DNA fragments (–128 to +52 of either wild type or mutant PaidB) generated by PCR using the primers 5′-aidBbam (5′-GGATCCGTGAAGATAACAC-3′) and 3′-aidB+52 (5′-AAAACGGTGTGAGTTTGCCAG-3′) were used for gel mobility shift assays, DNase I protection experiments, and KMnO4 reactivity assays. The template strand was labeled by 5′-phosphorylation of the 3′-aidB primer using phage T4 kinase and [γ-32P]ATP. Linear fragments of the csgB (238 bp) and sprE(P2) (248 bp) promoters were obtained by PCR using the primers B-H-csgB (5′-GGATCCAAGCTTGTCTGGTGCTTTTTGATAGCGG-3′), E-3b-csgB (5′-GAATTCATTT CAACTTGGTTGTTAACG-3′), E-sprE (5′-GAATTCGCTCCCAATGAGGAAAACC-3′), and B-sprE (5′-GGATCCTGGAAAGGAAAATGGACGAAC-3′). The mutant promoters harboring the C to T substitution at position –12 were generated using the double PCR method as described (40Yang C.H. Gavilanes-Ruiz M. Okinaka Y. Vedel R. Berthuy I. Boccara M. Chen J.W. Perna N.T. Keen N.T. Mol. Plant Microbe Interact. 2002; 15: 472-480Crossref PubMed Scopus (104) Google Scholar) and the above listed primers coupled either with 12AcsgB (5′-GGAAAGTAT a TCTGCGGAAAT-3′) and 12TcsgB (5′-ATTTCCGCAGA t ATACTTTCC-3′) (for csgB mutagenesis) or the primers 12TSprE (5′-ATAGCATGC t ACTATTGAGTA-3′) and 12ASprE (5′-TACTCAATAGT a GCATGCTAT-3′) (for sprE(P2) mutagenesis). Constructs were checked by sequencing. For gel mobility shift assays, the reconstituted holoenzyme (5–50 or 6–60 nm for the competition experiment) and the 180-bp promoter DNA fragments (1 nm) were incubated for 16 min at 37 °C in K-glu200 buffer in a final reaction volume of 10 μl. The reaction mixture was then loaded onto a native 5% polyacrylamide gel after the addition of 2.5 μl of loading buffer (50% sucrose, 0.025% xylene cyanol, 0.025% bromphenol blue, and 150 μg/ml heparin). For DNase I footprinting, reconstituted RNA polymerase (100 nm either EσS or Eσ70) was incubated with either PaidB(WT) or PaidB(12C→T) (4 nm) for 30 min at 37 °C in K-glu200 buffer. Protein-free DNA samples were treated with 1 μg/ml DNase I for 20 s, whereas the incubation was prolonged to 30 s in the presence of RNA polymerase. After the addition of loading buffer containing 150 μg/ml heparin, the samples were separated on a 5% native polyacrylamide gel, and the bands corresponding to the RNA polymerase-promoter complexes were eluted from the gel, precipitated, and re-suspended in EDTA (20 mm)-formamide buffer before being loaded on a 7% polyacrylamide sequencing gel. KMnO4 reactivity experiments were performed as described (41Colland F. Barth M. Hengge-Aronis R. Kolb A. EMBO J. 2000; 19: 3028-3037Crossref PubMed Google Scholar). Briefly, 50 nm RNA polymerase and promoters were incubated in K-glu200 (without dithiothreitol) for 15 min at 37 °C; KMnO4 was added to a final concentration of 10 mm, and the reaction was stopped after 30 s by adding 2-mercaptoethanol to a final concentration of 330 mm. For the kinetic reactivity experiment, samples were taken after 1.2, 2, 4, 6, 8, and 19 min of incubation. The KMnO4-reactive bands were expressed as a percentage of the total labeled DNA loaded onto the gel. Promoter Elements Determinant for Selective Recognition of the aidB Promoter by Eσ S—The aidB promoter (PaidB) is strongly σS-dependent in the absence of Ada activation, i.e. in physiological growth conditions (36Landini P. Hajec L.I. Volkert M.R. J. Bacteriol. 1994; 176: 6583-6589Crossref PubMed Google Scholar). To identify which features determine specific recognition of PaidB by EσS, we performed site-directed mutagenesis in and around the –10 sequence. We chose to target four putative promoter elements; as shown in Fig. 1A, a TG dinucleotide, typically a feature of σ70-dependent extended –10 promoters, is placed at an unusual location in PaidB (three and four nucleotides upstream of the –10 box instead of two and three). At this location, a TG motif cannot stimulate σ70-dependent transcription (28Burr T. Mitchell J. Kolb A. Minchin S. Busby S. Nucleic Acids Res. 2000; 28: 1864-1870Crossref PubMed Scopus (113) Google Scholar). We also targeted for mutagenesis the –13C residue, proposed to be the main feature for σS-specificity (7Espinosa-Urgel M. Chamizo C. Tormo A. Mol. Microbiol. 1996; 21: 657-659Crossref PubMed Scopus (103) Google Scholar, 8Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Crossref PubMed Google Scholar, 9Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and the –12C, i.e. the first nucleotide in the –10 hexamer. Finally, we changed to T the A nucleotide at position –6 immediately downstream of the –10 sequence, because T appears to be conserved at this position in σS-dependent promoters (8Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Crossref PubMed Google Scholar, 9Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). We examined the effect of the mutations on promoter activity in vivo using a low copy plasmid carrying the PaidB::lacZ transcriptional fusion (Fig. 1B). We compared promoter activity in the MV1161 strain (wild type relative to rpoS) and its rpoS derivative, MV2792, upon entry into stationary phase (OD600 nm ∼ 2.5). Substitutions that either disrupt the TG motif (15G→ C; designated G15C in Figs. 1, 2, 3, 4) or eliminate the –13C (13C→ G; designated C13G in Figs. 1, 2, 3, 4) show a rather small (1.5- to 2-fold) albeit significant and reproducible reduction in promoter activity, suggesting a possible role for these two promoter elements in modulation of affinity by EσS for PaidB. These effects are confirmed in a 15G→ C/13C→ G double mutant (Fig. 1B; this mutant is designated G15C/C13G in Figs. 1, 2, 3, 4). Substitution of the –6A nucleotide had no effect on transcription from PaidB. Displacement of the TG motif (15G→ T/14A→ G double mutant) to the optimal location for σ70 (–15/–14) slightly improves transcription by both EσS and Eσ70. Substitution of the –12C nucleotide to a T (12C→ T; designated C12T in Figs. 1, 2, 3, 4, 5, 6, 7) results in an almost perfect –10 sequence for σ70 (TATACT); the 12C→ T mutation strongly increases aidB transcription both in the wild type strain (roughly 4-fold) and the rpoS strain (20-fold). Although an increase of σ70-dependent transcription was expected for this promoter mutation, its extent is surprising; transcription from PaidBC12T reaches levels similar to the wild type aidB promoter in the wild type MV1161 strain. This observation suggests that the –12C plays a major role in selective recognition of the aidB promoter by EσS in vivo.Fig. 3Gel retardation assays in the presence of heparin. The formation of heparin-resistant complexes by EσS (A and C) and Eσ70 (B and D) is depicted. RNA polymerase concentrations used in the assay were 7.5, 15, and 50 nm. The σ/core RNA polymerase ratio was 2:1. PaidB-RNA polymerase complexes were separated on 5% native polyacrylamide gel and quantitated using a PhosphorImager. A typical experiment is shown in panels A (EσS)andB(Eσ70); the results in panels C and D are the average of three independent assays. Triangles (and dashed lines), wild type PaidB; diamonds, 15G→ C; circles, 13C→ G; boxes with internal intersecting lines, 15G→ C/13C→ G; boxes, 12C→ T. Open symbols indicate heparin-resistant complexes with the EσS form of RNA polymerase (C); closed symbols indicate heparin-resistant complexes with the Eσ70 form of RNA polymerase (D). WT, wild type; G15C, 15G→ C; C13G, 13C→ G; G15C/C13G, 15G→ C/13C→ G; C12T, 12C→ T; RNAP, RNA polymerase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4KMnO4 reactivity assay. A, KMnO4 reactivity of the –10 region on the template strand of either wild type (WT) or the different mutant aidB promoters (G15C, 15G→ C; C13G, 13C→ G; G15C/C13G, 15G→ C/13C→ G; C12T, 12C→ T). The positions of the reactive thymines are indicated. The arrow on the right indicates KMnO4 reactivity at the +2 position. C12T, 12C→ T. B, densitometric analysis of the reactivity of the +2T nucleotide expressed as percentage of total labeled DNA after correction (subtraction of densitometric value of the KMnO4-reactive band at +2 in the absence of RNA polymerase).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Kinetic reactivity to KMnO4 of the wild type and the 12C→ T mutant aidB promoters. Samples were incubated for 1.2, 2, 4, 6, and 19 min before treatment with KMnO4. A, reactivity of the –10 region of the template strand and position of the reactive thymine residues. WT, wild type; C12T, 12C→ T. B, densitometric analysis of the reactivity of the +2T nucleotide over time (between 0 and 6 min of incubation), performed as described in the Fig. 4 legend. White squares, wild type PaidB Eσ70; white triangles, wild type PaidB EσS; gray squares, PaidB(12C→ T) Eσ70; gray triangles, PaidB(C12T) EσS.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6DNase I protection experiment after heparin challenge. A, DNase I treatment was performed after 30 min of complex formation and followed by isolation of heparin-resistant complexes on a native acrylamide gel. Numbers at the left side indicate nucleotide positions according to the transcription start site as determined on the A + G sequencing reactions. The template strand is shown. DNase I-hypersensitive sites in the –35 and –50 areas are indicated by arrows. B and C, densitometric analysis of the gel shown in panel A between the –80 and –20 positions of either the wild type (WT) (B) or the 12C→ T (C12T) mutant PaidB (C). The dashed gray line indicates the reactivity of the unbound DNA. Black lines with closed symbols, EσS-PaidB complexes; gray lines with open symbols, Eσ70-PaidB complexes. The sequence of the template strand from position –80 to –20 is presented. The DNase I-hypersensitive sites showing differences between EσS and Eσ70 are highlighted by black letters. Underlined letters indicate every tenth nucleotide between position –20 and –80 and are given as a reference marker. Data are from a typical experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Competition experiment at the wild type and 12C→ T mutant promoters by EσS and Eσ70 forms of RNA polymerase. A, bandshift assay in the presence of heparin. The core enzyme and both sigma factors were reconstituted for 5 min at ratio 1:1:1 before complex formation. RNA polymerase concentrations used in the assay were 6, 18, 36, and 60 nm. Complexes with PaidB formed by either EσS or Eσ70 alone (60 nm) are shown on the right as reference. WT, wild type; C12T, 12C→ T. B, densitometric analysis of the gel shown in panel A. White boxes, wild type PaidB Eσ70; white triangles, wild type PaidB EσS; gray boxes, PaidB(C12T) Eσ70; gray triangles, PaidB(C12T), EσS; RNAP, RNA polymerase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Vitro Transcription Experiments—To confirm that the effects observed in vivo were indeed due to interaction between the promoter and the two forms of RNA polym
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