On the Role of the Escherichia coli RNA Polymerase σ70 Region 4.2 and α-Subunit C-terminal Domains in Promoter Complex Formation on the Extended –10 galP1 Promoter
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m304906200
ISSN1083-351X
AutoresLeonid Minakhin, Konstantin Severinov,
Tópico(s)RNA and protein synthesis mechanisms
ResumoBacterial promoters of the extended –10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity. Open promoter complexes can be formed on an extended –10 Escherichia coli galP1 promoter at temperatures as low as 6 °C, when complexes on most promoters are closed. Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended –10 motif and/or containing the –35 promoter consensus element. A panel of E. coli RNA polymerase holoenzymes containing two, one, or no α-subunit C-terminal domains (αCTD) and either wild-type σ70 subunit or σ70 lacking region 4.2 was assembled and tested for promoter complex formation. At 37 °C, αCTD and σ70 region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended –10 motif. However, no promoter complexes formed when both αCTD and σ70 region 4.2 were absent. Thus, in the context of an extended –10 promoter, αCTD and σ70 region 4.2 interactions with upstream DNA can functionally substitute for each other. In contrast, at low temperature, αCTD and σ70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for σ70 region 4.2 but not αCTD was required for open promoter complex formation on galP1 derivatives with extended –10 motif. We propose a model involving σ70 region 4.2 interaction with the β flap domain that explains these observations. Bacterial promoters of the extended –10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity. Open promoter complexes can be formed on an extended –10 Escherichia coli galP1 promoter at temperatures as low as 6 °C, when complexes on most promoters are closed. Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended –10 motif and/or containing the –35 promoter consensus element. A panel of E. coli RNA polymerase holoenzymes containing two, one, or no α-subunit C-terminal domains (αCTD) and either wild-type σ70 subunit or σ70 lacking region 4.2 was assembled and tested for promoter complex formation. At 37 °C, αCTD and σ70 region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended –10 motif. However, no promoter complexes formed when both αCTD and σ70 region 4.2 were absent. Thus, in the context of an extended –10 promoter, αCTD and σ70 region 4.2 interactions with upstream DNA can functionally substitute for each other. In contrast, at low temperature, αCTD and σ70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for σ70 region 4.2 but not αCTD was required for open promoter complex formation on galP1 derivatives with extended –10 motif. We propose a model involving σ70 region 4.2 interaction with the β flap domain that explains these observations. Most Escherichia coli promoters are characterized by the presence of two 6-bp sequence elements centered ∼10 and 35 nucleotides upstream of the transcription initiation point. These promoters are referred to as –10/–35 class promoters. Interaction of the RNA polymerase (RNAP) 1The abbreviations used are: RNAP, RNA polymerase; αCTD, α-subunit C-terminal domains; IPTG, isopropyl-1-thio-β-d-galactopyranoside.1The abbreviations used are: RNAP, RNA polymerase; αCTD, α-subunit C-terminal domains; IPTG, isopropyl-1-thio-β-d-galactopyranoside. σ70 subunit with –10 and –35 promoter elements is responsible for promoter recognition and transcription initiation. σ70 conserved region 4.2 recognizes the –35 promoter element, while σ70 conserved region 2.4 recognizes the –10 promoter element (reviewed by Gross et al., Ref. 1Gross C.A. Chan C. Dombroski A. Gruber T. Sharp M. Tupy J. Young B. Spring Harb. Symp. Quant. Biol. 1998; 63: 141-155Crossref PubMed Scopus (294) Google Scholar; see also Ref. 2Murakami K.S. Masuda S. Campbell E.A. Muzzin O. Darst S.A. Science. 2002; 296: 1285-1290Crossref PubMed Scopus (533) Google Scholar). Multiple alignments of promoter sequences permit the derivation of consensus sequences for the –10 and –35 promoter elements and show that most promoters deviate from the consensus (3Ozoline O.N. Tsyganov M.A. Nucleic Acids Res. 1995; 23: 4533-4541Crossref PubMed Scopus (46) Google Scholar). Promoter elements of strong promoters tend to deviate from consensus less than promoter elements of weak promoters. Thus, assuming that promoter elements with consensus sequence are preferred binding sites for σ70 regions 2.4 and 4.2, the strength of regions 2.4 and 4.2 interaction with their respective promoter elements determines the efficiency of promoter complex formation.For most promoters, RNAP σ70 regions 2.4 and 4.2 interactions with their target promoter elements are sufficient for promoter complex formation. On some promoters, the presence of RNAP α-subunit C-terminal domains (αCTDs) greatly increases transcription initiation beyond the basal level achieved through σ70-promoter element interactions (4Ross W. Gosink K.K. Salomon J. Igarashi K. Zou K. Ishihama A. Severinov K. Gourse R.L. Science. 1993; 262: 1407-1413Crossref PubMed Scopus (616) Google Scholar). On these promoters, αCTDs make sequence-specific interactions with an A-rich promoter element (the “UP-element”) located upstream of the –35 promoter element (reviewed by Gourse et al., Ref. 5Gourse R.L. Ross W. Gaal T. Mol. Microbiol. 2000; 37: 687-695Crossref PubMed Scopus (208) Google Scholar). In the absence of a UP-element, αCTD non-specifically interacts with upstream DNA and the stimulatory effect of these interactions is less significant.There exists a minor class of promoters that lack recognizable –35 promoter elements and whose –10 elements are extended with an upstream dinucleotide TG. Genetic data show that specific interaction between an additional region of σ70, conserved region 2.5, and the TG motif is required for promoter complex formation on promoters of this class (6Barne K.A. Bown J.A. Busby S.J. Minchin S.D. EMBO J. 1997; 16: 4034-4040Crossref PubMed Scopus (231) Google Scholar). Evidently, this additional contact is strong enough to make promoter complex formation on extended –10 promoters independent of σ70 region 4.2 and –35 promoter element interaction (7Kumar A. Malloch R.A. Fujita N. Smillie D.A. Ishihama A. Hayward R.S. J. Mol. Biol. 1993; 232: 406-418Crossref PubMed Scopus (208) Google Scholar).In order for template-directed RNA synthesis to occur, promoter DNA has to become locally melted (opened). In the catalytically competent open promoter complex, the melting extends from –12 to +3 positions and thus includes the entire –10 promoter element. Promoter opening is temperature-dependent, and promoter complexes formed on –10/–35 type promoters “close” below 15 °C (8Severinov K. Darst S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13481-13486Crossref PubMed Scopus (58) Google Scholar). In contrast, promoter complexes on the extended –10 galP1 promoter remain open at temperatures as low as 5 °C (9Grimes E. Busby S. Minchin S. Nucleic Acids Res. 1991; 19: 6113-6118Crossref PubMed Scopus (18) Google Scholar, 10Burns H.D. Minchin S.D. Nucleic Acids Res. 1994; 22: 3840-3845Crossref PubMed Scopus (31) Google Scholar, 11Attey A. Belyaeva T. Savery N. Hoggett J. Fujita N. Ishihama A. Busby S. Nucleic Acids Res. 1994; 22: 4375-4380Crossref PubMed Scopus (67) Google Scholar, 12Burns H.D. Belyaeva T.A. Busby S.J. Minchin S.D. Biochem. J. 1996; 317: 305-311Crossref PubMed Scopus (38) Google Scholar). The reason for this unusual behavior is not completely understood. For example, while it is clear that the extended –10 motif contributes to promoter opening at low temperature (9Grimes E. Busby S. Minchin S. Nucleic Acids Res. 1991; 19: 6113-6118Crossref PubMed Scopus (18) Google Scholar), it is not sufficient, since open promoter complexes on some extended –10 promoters are sensitive to low temperature (12Burns H.D. Belyaeva T.A. Busby S.J. Minchin S.D. Biochem. J. 1996; 317: 305-311Crossref PubMed Scopus (38) Google Scholar, 13Belyaeva T. Griffiths L. Minchin S. Cole J. Busby S. Biochem. J. 1993; 296: 851-857Crossref PubMed Scopus (43) Google Scholar).Previous work attempted to compare promoter complexes formed on the –10/–35 class and the extended –10 class promoters (9Grimes E. Busby S. Minchin S. Nucleic Acids Res. 1991; 19: 6113-6118Crossref PubMed Scopus (18) Google Scholar, 12Burns H.D. Belyaeva T.A. Busby S.J. Minchin S.D. Biochem. J. 1996; 317: 305-311Crossref PubMed Scopus (38) Google Scholar, 14Minchin S. Busby S. Biochem. J. 1993; 289: 771-775Crossref PubMed Scopus (23) Google Scholar). However, due to technical constrains, relatively large fragments of promoter DNA were altered. As a result, interpretation of some of the published data is complicated, since promoter complex formation can be affected by sequences outside of promoter consensus elements. In this work, we used several derivatives of the galP1 promoter obtained by site-specific mutagenesis and a set of RNAP mutants that lacked αCTD and/or σ70 region 4.2 to study promoter complexes formation in the absence of αCTD-UP element interactions, σ70 region 4.2- –35 promoter consensus element interactions and σ70 region 2.5-extended –10 motif interactions. Our results indicate that on the extended –10 galP1 promoter, αCTD-DNA interactions and σ70 region 4.2-DNA interactions are functionally equivalent, and at least one of these interactions is necessary for promoter complex formation at physiological temperature of 37 °C. In contrast, galP1 promoter complex formation at low temperature of 6 °C is strictly dependent on the presence of σ70 region 4.2.MATERIALS AND METHODSPlasmidsPlasmid pT7σ (15Zalenskaya K. Lee J. Gujuluva C.N. Shin Y.K. Slutsky M. Goldfarb A. Gene. 1990; 89: 7-12Crossref PubMed Scopus (58) Google Scholar) was used as a source of wild-type σ70. Plasmid pCYB2_σ1–565, expressing σ70-(1–565) fused to intein-chitin binding domain (CBD) was used as a source of σ70-(1–565) and is described by Severinov and Muir (16Severinov K. Muir T. J. Biol. Chem. 1998; 273: 16205-16209Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Ampicillin-resistant plasmid pREII-NHα45A-(1–235) expresses N-terminally hexahistidine-tagged α truncated at position 235 under the control of tandem lppP-lacPUV5 promoter (Niu et al., Ref. 17Niu W Kim Y. Tau G. Heyduk T. Ebright R. Cell. 1996; 87: 1123-1134Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Plasmids used to express RNAP core subunits are described elsewhere (18Tang H. Severinov K. Goldfarb A. Ebright R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4902-4906Crossref PubMed Scopus (110) Google Scholar). Plasmid pET21AsiA (19Severinova E. Severinov K. Darst S.A. J. Mol. Biol. 1998; 279: 9-18Crossref PubMed Scopus (79) Google Scholar) expresses C-terminally hexahistidine-tagged AsiA. Plasmid pTZ19galP1 containing the E. coli galP1 promoter was constructed as follows. The promoter-containing fragment was amplified from the pAA121 plasmid (19Severinova E. Severinov K. Darst S.A. J. Mol. Biol. 1998; 279: 9-18Crossref PubMed Scopus (79) Google Scholar) using PCR primers containing engineered EcoRI and HindIII recognition sites, respectively. The amplified fragment was treated with EcoRI and HindIII and cloned into appropriately treated pTZ19R. Plasmids harboring galP1 promoter derivatives were constructed by site-specific PCR mutagenesis using pTZ19galP1 as a template and cloned in pTZ19.Proteinsσ 70 and σ 70-(1–565)—The wild-type σ70 RNAP subunit was purified from BL21 (DE3) cells harboring the pT7σ plasmid as described (20Borukhov S. Goldfarb A. Protein Expr. Purif. 1993; 4: 503-511Crossref PubMed Scopus (103) Google Scholar).To purify C-terminally truncated σ70-(1–565), E. coli XL-1Blue cells were transformed with the pCYB2_σ1–565 plasmid. Transformants were grown in 1 liter of LB with ampicillin (100 μg/ml) at room temperature to an OD600 of 0.6–0.8, and expression was induced by the addition of IPTG to 1 mm. After 6 h, cells were harvested by centrifugation and resuspended in buffer H containing 20 mm HEPES, pH 8.0, 500 mm NaCl, 0.1 mm EDTA. Cells were lysed by sonication; the lysate was clarified by low speed centrifugation and loaded onto a 1-ml chitin column equilibrated in buffer H. The column was washed with 15 ml of buffer H and then quickly flushed with 3 column volumes of freshly prepared buffer H containing 30 mm dithiothreitol. The column outlet was sealed, and the column was left overnight at 4 °C. Pure σ70-(1–565) was eluted with 3 column volumes of buffer H without dithiothreitol, dialyzed against buffer H, and stored at –20 °C in the presence of 50% glycerol.RNAP Core Enzymes—The wild-type RNAP core enzyme and the mutant lacking both αCTDs were prepared by in vitro reconstitution as described (18Tang H. Severinov K. Goldfarb A. Ebright R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4902-4906Crossref PubMed Scopus (110) Google Scholar, 20Borukhov S. Goldfarb A. Protein Expr. Purif. 1993; 4: 503-511Crossref PubMed Scopus (103) Google Scholar). The core and holoenzyme fractions were separated on a 1-ml Protein-Pak Q 8 HR column (Waters) attached to a Waters 650 FPLC. RNAP was loaded on the column in TGE buffer (10 mm Tris-HCl, pH 7.9, 1 mm EDTA, 5% glycerol) and eluted using a linear 60-ml gradient of NaCl from 0.23 to 0.4 m. Fractions containing RNAP core and holoenzymes were pooled separately, concentrated to 1–2 mg/ml and stored at –20 °C in the presence of 50% glycerol. Heterodimeric RNAP core enzyme containing one wild-type α-subunit and one α-subunit lacking CTD was purified from XL1-Blue strain transformed with the plasmid pREII-NHα45A (1–235) according to the procedure of Niu and co-workers (21Estrem S.T. Ross W. Gaal T. Chen Z.W. Niu W. Ebright R.H. Gourse R.L. Genes Dev. 1999; 13: 2134-2147Crossref PubMed Scopus (162) Google Scholar). The core and holoenzyme fractions were separated on the Protein-Pak Q8 HR column (Waters) and stored as described above.AsiA—E. coli BL21 (DE3) cells containing the pLysE plasmid (Novagen) were transformed with pET21AsiA. Transformants were grown in 2 liters of LB with ampicillin (100 μg/ml) and chloramphenicol (25 μg/ml) at room temperature to OD600 of 0.6–0.8 and expression was induced by the addition of IPTG to 1 mm. After 6 h, cells were harvested by centrifugation and resuspended in buffer C containing 20 mm Tris-HCl, pH 7.9, 5% glycerol, 500 mm NaCl. Cells were lysed by sonication and after a low speed centrifugation, cell extract was loaded onto a 5-ml chelating Sepharose column (Amersham Biosciences) loaded with Ni2+ and attached to an FPLC. The column was washed with buffer C containing 20 mm imidazole, and AsiA was eluted with 100 mm imidazole in the buffer. The eluate was diluted 5-fold with buffer D (50 mm Tris-HCl, pH 7.9, 5% glycerol, 50 mm NaCl, 1.65 m (NH4)2SO4, 1 mm β-mercaptoethanol) and loaded onto a 8 ml phenyl-Toyopearl column equilibrated with the same buffer. The column was washed and eluted with an (NH4)2SO4 gradient from 1.3 to 0 m. Homogeneous AsiAHIS protein was eluted from the column at 20–0 mm (NH4)2SO4, concentrated to 1 mg/ml concentration and stored at –20 °C in the presence of 50% glycerol.In Vitro TranscriptionAbortive transcription initiation reactions contained, in 10 μl of transcription buffer (50 mm Tris-HCI, pH 7.9, 10 mm MgCl2, 40 mm KCl), 20 nm RNAP core enzyme and 320 nm σ70 or 640 nm σ70-(1–565). Reactions were incubated for 10 min at 37 °C, followed by the addition of 10 nm promoter fragments, 0.1 mm initiating dinucleotide CpA, and 3 μCi (3000 Ci/mmol) of [α-32P]UTP. Reactions proceeded for 10 min at 37 °C and were terminated by the addition of an equal volume of loading buffer containing 9 m urea. Reaction products were resolved by electrophoresis in denaturing (8 m urea) 20% (19:1) polyacrylamide gel, visualized by autoradiography, and quantified using the Molecular Dynamics PhosphorImager.Determination of Open Complex Dissociation KineticsOpen complex lifetime was measured on linear promoter-containing DNA fragments using an abortive transcription assay. Reactions contained, in 40 μl of buffer (40 mm Tris-HCl, pH 7.9, 10 mm MgCl2, 175 mm NaCl, 2 mm β–mercaptoethanol), 80 nm wild-type and mutant RNAP core enzymes and 80 nm σ70 or 160 nm σ565. Reactions were incubated for 10 min at 30 °C, followed by the addition of 10 nm promoter fragments and additional incubation for 15 min at 30 °C. A 10-μl reaction aliquot was transferred to tubes containing 0.1 mm dinucleotide CpA, 10 μm UTP, and 3 μCi (3000 Ci/mmol) of [α-32P]UTP. Abortive transcription initiation was allowed to proceed for 10 min and was terminated by the addition of an equal volume of loading buffer containing 9 m urea. The remainder of promoter complex reaction was supplemented with 100 μg/ml heparin and incubation at 30 °C was continued. 10-μl reaction aliquots were withdrawn immediately after the heparin addition and 30 and 90 min after the heparin addition and assayed for abortive transcription as described above. Reaction products were resolved, visualized, and quantified as described above.DNase I FootprintingThe 147-bp DNA fragments (–96 to +51) harboring the galP1 promoter and its derivatives were amplified by PCR from appropriate plasmids using universal T7 promoter and M13 reverse primers. PCR fragments were digested with HindIII and labeled at nontemplate strand by filling the HindIII sticky end with Klenow enzyme in the presence of [α-32P]dCTP. Promoter complexes were formed in 15 μl of transcription buffer containing 200 nm RNAP core enzymes and a 4-fold excess of σ70 or 8-fold excess of σ70-(1–565). Reactions were incubated for 10 min at 37 °C, followed, when necessary, by the addition of 1 μm of AsiA and further 10 min incubation at the same temperature. Reactions were next supplemented with 10 nm32P-endlabeled promoter fragments and incubated for 20 min at assay temperature. DNase I footprinting reactions were initiated by the addition of 0.1 or 1 unit of DNase I (Worthington) for 37 and 6 °C reactions, respectively. The reactions proceeded for 30 s at the assay temperature and were terminated by addition of 85 μl stop-mixture (20 mm EDTA, 10 μg of denatured calf thymus DNA, and water) followed by phenol extraction and ethanol precipitation. The DNA samples were dissolved in 8 μl of formamide-loading dye and analyzed using 7% polyacrylamide/8 m urea sequencing gels.KMnO4 ProbingReactions were set up as described above for DNase I footprinting. Promoter complexes were treated with 1 mm KMnO4 for 15 s at the assay temperature. Reactions were terminated by the addition of β-mercaptoethanol to 300 mm, followed by phenol extraction, ethanol precipitation, and 20 min treatment with 10% piperidine at 95 °C. Reaction products were analyzed using 7% polyacrylamide urea gels.Gel Retardation AssayThe reactions contained, in 20 μl of transcription buffer, 10 nm RNAP wild-type and mutant core enzymes combined with appropriate amounts of σ70 or σ70-(1–565). Reactions were incubated for 15 min at 37 °C, transferred to 6 °C, and incubated for additional 10 min, followed by the addition of 10 nm32P-endlabeled galP1 promoter fragment. After further 30 min of incubation at 6 °C, reactions were combined with 4 μl of loading buffer (transcription buffer containing 50% glycerol and 0.05% bromphenol blue). When necessary, heparin was added to the final concentration 50 μg/ml, and reactions were immediately loaded on 5% (29:1) polyacrylamide Tris borate/EDTA gel. The gel was run in a cold room and reactions products were revealed by autoradiography.RESULTSThe galP1 Promoter Derivatives Used in This Work—A galP1 promoter derivative previously constructed and characterized by Burns et al. (12Burns H.D. Belyaeva T.A. Busby S.J. Minchin S.D. Biochem. J. 1996; 317: 305-311Crossref PubMed Scopus (38) Google Scholar) was used as a starting point of this work. The promoter differs from the natural galP1 promoter by three point mutations. Two substitutions at positions –9 and –8 relative to the start point of transcription create a consensus extended –10 promoter element, TGcTATAAT, instead of the wild-type sequence TGcTATggT. The third substitution introduces a T at position –19 and destroys the overlapping galP2 promoter, thus simplifying the analysis of galP1 promoter complexes. We used site-directed mutagenesis to construct “second-generation” derivatives of the optimized galP1 promoter constructed by Burns et al. (Fig. 1A). The first derivative, galP1-35, contains the consensus –35 promoter element, TTGACA, incorporated 18 base pairs upstream of T at position –12, the first base of the –10 consensus element. The second derivative, galP1-TG, was constructed by substituting the TG motif of the extended –10 promoter consensus element for AC. The resultant promoter was named galP1-TG. The final construct, galP1-35-TG, is a derivative of galP1-35 and also has the TG extended –10 motif substituted with AC. Note that our nomenclature differs from that adopted by Kamali-Moghaddam and Geiduschek in the accompanying article (28Kamali-Moghaddam M. Geiduschek E.P. J. Biol. Chem. 2003; 278: 29701-29709Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar).The ability of promoter fragments containing galP1 and its derivatives to serve as templates for the synthesis of CpApU from the CpA primer and the UTP substrate by E. coli RNAP σ70 holoenzyme was investigated (Fig. 1B). DNA fragments containing galP1, galP1-35 as well as galP1-35-TG supported high and comparable levels of CpApU synthesis. In contrast, little transcription was detected when RNAP was combined with the galP1-TG fragment (less than 5% compared with other promoters used here). These result indicate the following. (i) galP1 has no functional –35 promoter consensus element. (ii) The –10 consensus promoter element alone, in the absence of the TG motif, is insufficient for transcription initiation, and (iii) the consensus –35 element sequence introduced in galP1-35-TG promoter is functional.Promoter Complex Formation by RNAP Mutants Lacking αCTD and/or σ 70 Region 4.2 on galP1 Derivatives—We wished to determine the contribution of RNAP domains capable of interaction with upstream promoter sequences, the αCTD and the σ70 subunit region 4.2, to promoter complex formation on galP1 and its derivatives. To this end, 5 mutant RNAP holoenzymes that lack one or both αCTDs and/or σ70 region 4.2 and wild-type σ70 RNAP holoenzyme were prepared. RNAP holoenzymes were reconstituted in vitro by combining RNAP core containing two copies of the wild-type α subunit, RNAP core that has one α and another copy truncated at amino acid 235, and RNAP core that contains two copies of truncated α with either wild-type σ70, or with a σ70 mutant that is truncated at amino acid position 565, σ70-(1–565), and thus lacks region 4.2 (16Severinov K. Muir T. J. Biol. Chem. 1998; 273: 16205-16209Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 22Minakhin L. Niedziela-Majka A. Kuznedelov K. Adelman K. Heyduk T. Severinov K. J. Mol. Biol. 2003; 326: 679-690Crossref PubMed Scopus (24) Google Scholar). Native PAGE analysis showed that reconstituted RNAP holoenzymes contained less than 5% RNAP core (data not shown). The ability of mutant holoenzymes and wild-type RNAP control to form promoter complexes at 37 °C with DNA fragments containing galP1 and its active derivatives was investigated by DNase I footprinting. The footprinting results obtained with DNA fragments that contained functional promoters galP1, galP1-35, and galP1-35-TG are presented in Fig. 2, A–C, respectively. On all three promoters, the wild-type holoenzyme protected DNA from +20 to –23 (Fig. 2, A–C, lane 2). DNase I hypersensitive sites centered at positions –25, –36, –44, –57, and –67 were also observed in complexes formed on all three promoters with wild-type RNAP. The DNA between the hypersensitive sites was protected (Fig. 2, A–C, lane 2). This periodic pattern of protection and hypersensitivity has been attributed to wrapping of upstream DNA around RNAP (Attey et al., Ref. 11Attey A. Belyaeva T. Savery N. Hoggett J. Fujita N. Ishihama A. Busby S. Nucleic Acids Res. 1994; 22: 4375-4380Crossref PubMed Scopus (67) Google Scholar). The hypersensitive site at position –25, was very prominent in the galP1-35 and galP1-35-TG complexes, and was less pronounced in galP1 complexes. DNase I hypersensitivity at about –25 is a common feature of promoter complexes formed by bacterial RNAP (3Ozoline O.N. Tsyganov M.A. Nucleic Acids Res. 1995; 23: 4533-4541Crossref PubMed Scopus (46) Google Scholar); it may result from DNA bending that arises when RNAP establishes simultaneous contacts with the –35 and –10 promoter consensus elements (2Murakami K.S. Masuda S. Campbell E.A. Muzzin O. Darst S.A. Science. 2002; 296: 1285-1290Crossref PubMed Scopus (533) Google Scholar). If this interpretation is correct, weaker hypersensitivity in the galP1 complexes is expected, since σ70 region 4.2 does not make specific contacts with DNA on galP1. In the galP1-35, and galP1-35-TG complexes, but not in galP1 complexes, position –5 was exposed to DNase I attack (compare, for example Fig. 2, A–C, lane 2).Fig. 2Promoter complex formation on galP1 promoter derivatives using mutant RNAP holoenzymes lacking αCTD and/or σ70 region 4.2. Promoter complexes were formed at 37 °C using the indicated RNAP holoenzymes on DNA fragments containing indicated functional galP1 promoter derivatives. Promoter fragments were 32P-endlabeled at the top (non-template strand). Promoter complexes were footprinted with DNase I, reaction products were separated by electrophoresis in a 7% sequencing gel and revealed by autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)On all three promoters, complexes formed by σ70 holoenzymes lacking one of the αCTDs were very similar to the corresponding wild-type RNAP complexes, though protection of a DNase I-sensitive band at –52 was decreased somewhat (Fig. 2, A–C, compare lanes 2 and 3). In complexes formed by σ70 holoenzyme lacking both αCTDs, protection at –52 and –48 was further decreased (Fig. 2, A–C, compare lane 4 with lanes 3 and 2), suggesting that in the wild-type RNAP complexes, protection of AT-rich sequence between positions –52 and –48 is due to αCTD binding, in agreement with earlier results (11Attey A. Belyaeva T. Savery N. Hoggett J. Fujita N. Ishihama A. Busby S. Nucleic Acids Res. 1994; 22: 4375-4380Crossref PubMed Scopus (67) Google Scholar).No footprint was observed when σ70-(1–565) holoenzymes were combined with galP1-35-TG (Fig. 2C, lanes 5–7). This result is expected, since the interaction between σ70 region 4.2 and the –35 promoter consensus element is essential for promoter complex formation on the –10/–35 class promoters. In contrast, footprints were readily observed when σ70-(1–565) holoenzymes reconstituted from wild-type core enzyme or from core enzyme lacking one αCTD were combined with galP1 and galP1-35, also as expected (Fig. 2, A and B, lanes 5 and 6). These footprints are distinct from the corresponding σ70 holoenzyme footprints in that position –37, which is fully protected in the presence of σ70 RNAP complexes, is hypersensitive in the presence of σ70-(1–565) RNAP, presumably because specific interactions between σ70 region 4.2 and the –35 promoter element (galP1-35) or nonspecific interactions between σ70 region 4.2 and promoter DNA (galP1) are lacking. In complexes formed by σ70-(1–565) RNAP holoenzymes containing both αCTDs, protection at around –40/–50 is present (Fig. 2, A and B, lane 5), suggesting that αCTD is able to interact with its binding site independently of σ70 region 4.2. Hypersensitivity at –67 and at –57 is absent in σ70-(1–565) complexes. The reasons for this are unclear, since σ70 region 4.2 is only expected to interact with promoter DNA ∼35 base pairs upstream of the transcription start point (1Gross C.A. Chan C. Dombroski A. Gruber T. Sharp M. Tupy J. Young B. Spring Harb. Symp. Quant. Biol. 1998; 63: 141-155Crossref PubMed Scopus (294) Google Scholar).Surprisingly, RNAP holoenzyme lacking both αCTDs and σ70 region 4.2 produced no footprints on extended –10 galP1 or galP1-35 promoters (Fig. 2, A and B, lane 7). Since the removal of these RNAP domains individually had no effect on promoter complex formation, we conclude that on galP1 and galP1-35, αCTD, and σ70 region 4.2 interactions with upstream promoter DNA can substitute for each other. However, the removal of both of these interactions prevents promoter complex formation even in the context of a consensus extended –10 promoter.Stability of Promoter Complexes—The dissociation kinetics of promoter complexes formed on galP1 and its two active derivatives, galP1-35 and galP1-35-TG, was investigated. Promoter complexes were formed, challenged with DNA competitor heparin, and the amount of complexes that survived heparin challenge was determined by withdrawing reaction aliquots at various times after heparin addition and supplementing them with the CpA primer and radioactive UTP substrate to allow the production of radioactively labeled abortive transcript CpApU. Reaction products were separated by denaturing PAGE and the decline of CpApU synthesis over time was monitored. The results are presented in Fig. 3A. As can be seen, in the case of the wild-type RNAP promoter complexes on galP1-35, no decline in abortive synthesis was observed even after 90-min incubation in the presence of heparin. Complexes on galP1-35-TG we
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