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• W2137767952 abstract "At the spoIIG promoter phosphorylated Spo0A (Spo0A∼P) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation. We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters. Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0A∼P, but heteroduplexes with 4 or 2 base pairs denatured required Spo0A∼P for maximal levels of transcription. Investigation of the thermal dependence of transcription suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A∼P does not reduce this energetic barrier. Kinetic assays revealed that Spo0A∼P stimulated both the rate of formation of initiated complexes as well as increasing the number of complexes capable of initiating transcription. These results imply that Spo0A∼P stimulates transcription at least in part by stabilizing the RNA polymerase-spoIIG complex until contacts between RNA polymerase and the -10 element induce strand separation. At the spoIIG promoter phosphorylated Spo0A (Spo0A∼P) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation. We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters. Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0A∼P, but heteroduplexes with 4 or 2 base pairs denatured required Spo0A∼P for maximal levels of transcription. Investigation of the thermal dependence of transcription suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A∼P does not reduce this energetic barrier. Kinetic assays revealed that Spo0A∼P stimulated both the rate of formation of initiated complexes as well as increasing the number of complexes capable of initiating transcription. These results imply that Spo0A∼P stimulates transcription at least in part by stabilizing the RNA polymerase-spoIIG complex until contacts between RNA polymerase and the -10 element induce strand separation. Transcription initiation is an intricate process accomplished by the interplay between promoter DNA and a multisubunit RNA polymerase core enzyme, α2ββ′, complexed with one of several alternative sigma factors, σ. In Bacillus subtilis, the majority of transcripts during logarithmic growth are produced by RNA polymerase complexed with σA, a homolog of the Escherichia coli sigma factor, σ70. Promoters used by such RNA polymerase (RNAP) 1The abbreviations used are: RNAP, RNA polymerase; Spo0A∼P, phosphorylated Spo0A. 1The abbreviations used are: RNAP, RNA polymerase; Spo0A∼P, phosphorylated Spo0A. holoenzymes contain two conserved sequence elements centered at -35 and -10 (relative to the start site of transcription) separated by an optimal distance of 17 bp (1Gross C.A. Chan C. Dombroski A. Gruber T. Sharp M. Tupy J. Young B. Cold Spring Harb. Symp. Quant. Biol. 1998; 63: 141-155Crossref PubMed Scopus (294) Google Scholar). Additional nearby sequence elements may also contribute to the kinetics of transcription initiation (2Ross W. Gosink K.K. Salomon J. Igarashi K. Zou C. Ishihama A. Severinov K. Gourse R.L. Science. 1993; 262: 1407-1413Crossref PubMed Scopus (616) Google Scholar, 3Keilty S. Rosenberg M. J. Biol. Chem. 1987; 262: 6389-6395Abstract Full Text PDF PubMed Google Scholar). RNAP-promoter complexes pass through several intermediates during initiation including closed complexes in which the DNA is fully double-stranded and open complexes in which the DNA strands are partially separated as a prelude to RNA polymerization (4Record Jr., M.T. Reznikoff W.S. Craig M.L. McQuade K.L. Schlax P.J. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. Vol. I. American Society for Microbiology, Washington, D.C.1996: 792-821Google Scholar). The physical basis for DNA strand separation remains poorly understood. The exact sequence of promoter elements and their architecture dictates RNAP occupancy at each promoter and the attendant isomerization rates between each of the intermediates and so sets the intrinsic level of transcription initiation. Transcription factors are thought to compensate for suboptimal sequence or spacing of promoter elements whose regulatory task it is to allow specific promoters to respond under appropriate conditions at appropriate rates. Some transcription factors, best exemplified by catabolite activator protein at the lac promoter in E. coli, interact with the α subunit of RNAP to recruit the holoenzyme, increasing the number of RNAP-promoter complexes formed and therefore the amount of transcripts produced (Ref. 5Busby S. Ebright R.H. J. Mol. Biol. 1999; 293: 199-213Crossref PubMed Scopus (628) Google Scholar, but see Ref. 6Liu M. Gupte G. Roy S. Bandwar R.P. Patel S.S. Garges S. J. Biol. Chem. 2003; 278: 39755-39761Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Others, the canonical example being λ cI at PRM, facilitate open complex formation through contacts with the σ subunit of RNAP without stimulating holoenzyme binding (7Malan T.P. Kolb A. Buc H. McClure W.R. J. Mol. Biol. 1984; 180: 881-909Crossref PubMed Scopus (157) Google Scholar, 8Hawley D.K. McClure W.R. J. Mol. Biol. 1982; 157: 493-525Crossref PubMed Scopus (163) Google Scholar, 9Kuldell N. Hochschild A. J. Bacteriol. 1994; 176: 2991-2998Crossref PubMed Google Scholar, 10Li M. Moyle H. Susskind M.M. Science. 1994; 263: 75-77Crossref PubMed Scopus (141) Google Scholar). It has been proposed that λ cI might facilitate initiation by counteracting the tendency of σ to disengage from the -35 element during isomerization (11Dove S.L. Huang F.W. Hochschild A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13215-13220Crossref PubMed Scopus (44) Google Scholar). We have studied the effects of Spo0A∼P at the σA-dependent promoter of the spoIIG operon, which encodes σE, one of the first of the sporulation-specific sigma factors in B. subtilis, as representative of the class of transcription factors that facilitate open complex formation without recruiting RNAP. Spo0A is a response regulator controlling a genetic network that governs commitment to sporulation in B. subtilis (12Stragier P. Losick R. Annu. Rev. Genet. 1996; 30: 297-341Crossref PubMed Scopus (510) Google Scholar). It lies at the terminus of a phosphorelay, an extended two-component signal transduction system whose complexity is indicative of the diversity of signals that must be integrated prior to the activation of this protein (13Burbulys D. Trach K.A. Hoch J.A. Cell. 1991; 64: 545-552Abstract Full Text PDF PubMed Scopus (650) Google Scholar, 14Ohlsen K.L. Grimsley J.K. Hoch J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1756-1760Crossref PubMed Scopus (129) Google Scholar, 15Perego M. Hanstein C. Welsh K.M. Djavakhishvili T. Glaser P. Hoch J.A. Cell. 1994; 79: 1047-1055Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 16Perego M. Hoch J.A. Trends Genet. 1996; 12: 97-101Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 17Perego M. Glaser P. Hoch J.A. Mol. Microbiol. 1996; 19: 1151-1157Crossref PubMed Scopus (113) Google Scholar). Like most other response regulators, Spo0A is a transcription factor whose activity is modulated by reversible phosphorylation of an aspartate residue within the highly conserved N-terminal receiver domain (18Spiegelman G.B. Bird T.H. Voon V. Hoch J.A. Silhavy T.J. Two-Component Signal Transduction. American Society for Microbiology, Washington, D.C.1995: 159-179Google Scholar, 19Hoch J.A. Silhavy T.J. Hoch J.A. Silhavy T.J. Two-Component Signal Transduction. American Society for Microbiology, Washington, D.C.1995Crossref Google Scholar). Although not well understood mechanistically, phosphorylation of this module enhances the affinity of the protein for its DNA-binding site, the 0A box, 5′-TGTCGAA-3′ (20Baldus J.M. Green B.D. Youngman P. Moran Jr., C.P. J. Bacteriol. 1994; 176: 296-306Crossref PubMed Google Scholar, 21Strauch M. Webb V. Spiegelman G. Hoch J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1801-1805Crossref PubMed Scopus (248) Google Scholar). Improved DNA binding permits transcription modulation by the C-terminal domain, which may either activate or repress transcription depending on the orientation and position of the 0A boxes (18Spiegelman G.B. Bird T.H. Voon V. Hoch J.A. Silhavy T.J. Two-Component Signal Transduction. American Society for Microbiology, Washington, D.C.1995: 159-179Google Scholar). At the spoIIG promoter, Spo0A∼P stimulates transcription (20Baldus J.M. Green B.D. Youngman P. Moran Jr., C.P. J. Bacteriol. 1994; 176: 296-306Crossref PubMed Google Scholar, 22Bird T.H. Grimsley J.K. Hoch J.A. Spiegelman G.B. Mol. Microbiol. 1993; 9: 741-749Crossref PubMed Scopus (47) Google Scholar). The spacing of the spoIIG promoter elements is unusual; the transcription start site is located 2 bp further downstream from the -10 element than at an ideal promoter, and 22 bp instead of the optimal 17 separate the -35 and -10 elements. The latter characteristic forms the basis of the requirement for activated Spo0A to stimulate transcription (23Kenney T.J. York K. Youngman P. Moran Jr., C.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9109-9113Crossref PubMed Scopus (65) Google Scholar). In vitro RNAP binds readily, albeit weakly, to this promoter but on linear templates requires Spo0A∼P to initiate efficiently (22Bird T.H. Grimsley J.K. Hoch J.A. Spiegelman G.B. Mol. Microbiol. 1993; 9: 741-749Crossref PubMed Scopus (47) Google Scholar, 24Bird T.H. Grimsley J.K. Hoch J.A. Spiegelman G.B. J. Mol. Biol. 1996; 256: 436-448Crossref PubMed Scopus (35) Google Scholar). Normal regulation appears to be a consequence of Spo0A∼P binding to a pair of 0A boxes located between -53 and -37 relative to the start site of transcription (+1). The promoter proximal of these boxes completely overlaps the -35 element used by RNAP, and the contacts between Spo0A and the σA subunit of RNAP necessary to stimulate transcription from this promoter have been defined (25Hatt J.K. Youngman P. J. Bacteriol. 1998; 180: 3584-3591Crossref PubMed Google Scholar, 26Baldus J.M. Buckner C.M. Moran Jr., C.P. Mol. Microbiol. 1995; 17: 281-290Crossref PubMed Scopus (36) Google Scholar, 27Buckner C.M. Schyns G. Moran Jr., C.P. J. Bacteriol. 1998; 180: 3578-3583Crossref PubMed Google Scholar, 28Schyns G. Buckner C.M. Moran Jr., C.P. J. Bacteriol. 1997; 179: 5605-5608Crossref PubMed Google Scholar). Kinetic analysis of in vitro transcription initiation has shown that Spo0A∼P increases the overall transcription rate by increasing the rate of isomerization of RNAP-promoter complexes to the initiated state but has no apparent effect in recruiting RNAP to the promoter (24Bird T.H. Grimsley J.K. Hoch J.A. Spiegelman G.B. J. Mol. Biol. 1996; 256: 436-448Crossref PubMed Scopus (35) Google Scholar). Moreover, structural studies have found that the addition of Spo0A∼P and RNAP induces DNA strand separation between -14 and -3 at the wild type spoIIG promoter and that the activator requirement can be bypassed by artificial strand separation using heteroduplex templates implying that Spo0A∼P cooperates with RNAP to denature the DNA (29Rowe-Magnus D.A. Spiegelman G.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5305-5310Crossref PubMed Scopus (20) Google Scholar). In this communication we have used an in vitro transcription assay to compare the effects of Spo0A∼P on the temperature dependence and the rate of initiated complex formation using an extended series of heteroduplex templates with decreasing numbers of denatured bases. The data indicate that the thermodynamic properties of initiation are driven by transitions involving only RNAP and the DNA and that Spo0A∼P stimulates the rate at which RNAP establishes interactions with the nontemplate strand of the -10 element. Heteroduplex Construction—Heteroduplex templates were created as described earlier (29Rowe-Magnus D.A. Spiegelman G.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5305-5310Crossref PubMed Scopus (20) Google Scholar). A PCR product was generated using Taq polymerase (Invitrogen), the template pUCIIGtrpA (22Bird T.H. Grimsley J.K. Hoch J.A. Spiegelman G.B. Mol. Microbiol. 1993; 9: 741-749Crossref PubMed Scopus (47) Google Scholar), a mutagenic primer complementary to the nontemplate strand, SS3 (5′-CAGAGCTTGCTTATATCTTATGAAGCAAGAAGGGG-3′; purchased from the Nucleic Acid and Protein Service Unit, University of British Columbia), and a downstream primer (IIG2), which anneals to the coding strand adjacent to the BamHI site. The primer SS3 contained nucleotides that were identical to the template strand from -14 to -11. The PCR product was cloned into the pGEM-T vector (Promega) to create pSS3, and the inserts were verified by sequencing. The HindIII-AluI fragment was isolated from pUCIIGtrpA and ligated to the AluI-BamHI fragments from pSS3, and the ligation mixture was used as a template for PCR by using IIG2 and an upstream primer, IIGA, which anneals to the noncoding strand adjacent to the HindIII site. The resulting product was ligated into pGEM-T to create pSS3IIG. The E. coli strain DH5α was used for all transformations, and plasmid preparations were performed as described by Sambrook et al. (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The plasmid pSS3IIG was digested with HindIII and BamHI, and the promoter-bearing fragment ligated into pBluescript SK+ vector (Stratagene) was digested with the same enzyme to create pSK3IIG+. The wild type spoIIG promoter had previously been cloned into pBluescript SK+ and pBluescript SK- to create pSKIIG+ and pSKIIG- (29Rowe-Magnus D.A. Spiegelman G.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5305-5310Crossref PubMed Scopus (20) Google Scholar). The pSKIIG+ plasmid was used as the template in a PCR to create pSK6IIG+ and pSK7IIG+. The reactions composed of primer pairs, IIG6NT (5′-CCCACAGAGCTTGCTTATATGATATGAAGCAAGAAGGG-3′) and IIG6T (5′-CCCTTCTTGCTTCATATCATATAAGCAAGCTCTGTGGG-3′) or IIG7NT (5′-CCCACAGAGCTTGCTTATTACTTATGAAGCAAGAAGGG-3′) and IIG7T (5′-CCCTTCTTGCTTCATAAGTAATAAGCAAGCTCTGTGGG-3′), along with 2.5 mm dNTPs, 1× Pfu Turbo buffer, and 1 unit of Pfu (Stratagene) were subjected to 18 cycles of 30 s at 95 °C, 1 min at 55 °C, and 6 min at 68 °C. The PCR products were treated with two successive additions of 10 units of DpnI and ethanol-precipitated after the addition of 0.5 μg of salmon sperm DNA. The DNA was resuspended in water and used to transform E. coli DH5α. Mutations in transformants were verified by sequencing. Single-stranded DNA was produced and purified, and the heteroduplex templates were generated as described earlier (29Rowe-Magnus D.A. Spiegelman G.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5305-5310Crossref PubMed Scopus (20) Google Scholar). The templates are named to reflect the fact that they contain the DNA sequence from the nontemplate strand on both stands in the single-stranded region. The name also indicates the boundaries of the single-stranded region (see Fig. 1). For consistency, templates MB12NT and MB8NT that were described earlier were renamed NT14/3 and NT14/7, respectively. Template DNA—The spoIIG and IIG17 templates were isolated from the plasmids pUCIIGtrpA and pUCIIG17trpA, respectively, as 415-bp fragments generated by digestion with PvuII and BamHI. These fragments contained the promoter and both upstream Spo0A-binding sites but lacked the trpA terminator resulting in run-off transcripts of ∼130 bp. The fragments were isolated by electrophoresis, recovered using a QIAquick gel extraction kit (Qiagen), and stored in 10 mm HEPES (pH 7.9), 20 mm potassium acetate, and 0.1 mm EDTA at 4 °C. The concentration of template fragments was determined by measuring absorbance at 260 nm. After annealing the single-stranded DNA, heteroduplex templates were treated in an identical fashion. The plasmid pUCIIG17trpA contains a modified promoter in which 5 bp have been deleted between the -35 and -10 consensus elements (see Fig. 1). 2B. McLeod and G. B. Spiegelman, unpublished observation. In Vitro Transcription Assays—RNAP was prepared from mid-log phase B. subtilis cells as described by Dobinson and Spiegelman (31Dobinson K.F. Spiegelman G.B. Biochemistry. 1987; 26: 8206-8213Crossref PubMed Scopus (38) Google Scholar). Spo0A and phosphorelay proteins were prepared as previously described (32Zhou X.Z. Madhusudan Whiteley J.M. Hoch J.A. Varughese K.I. Proteins. 1997; 27: 597-600Crossref PubMed Scopus (13) Google Scholar, 33Zapf J.W. Hoch J.A. Whiteley J.M. Biochemistry. 1996; 35: 2926-2933Crossref PubMed Scopus (50) Google Scholar, 34Grimshaw C.E. Huang S. Hanstein C.G. Strauch M.A. Burbulys D. Wang L. Hoch J.A. Whiteley J.M. Biochemistry. 1998; 37: 1365-1375Crossref PubMed Scopus (88) Google Scholar), as was the activation of Spo0A (22Bird T.H. Grimsley J.K. Hoch J.A. Spiegelman G.B. Mol. Microbiol. 1993; 9: 741-749Crossref PubMed Scopus (47) Google Scholar). Transcription assays were carried out by composing 8 μl of an initiation mix that contains: 1 μl of 10× transcription buffer (24Bird T.H. Grimsley J.K. Hoch J.A. Spiegelman G.B. J. Mol. Biol. 1996; 256: 436-448Crossref PubMed Scopus (35) Google Scholar), 2 μl of 20 nm template DNA, 2 μlof4mm Spo0A∼P, 600 nm ATP, 50 μm GTP, and 3 μCi of [α32P]GTP (800 Ci/mmol; PerkinElmer Life Sciences). The reaction tubes were incubated at the indicated temperature for 90 s before the addition of 1 μl of RNAP. After the indicated preincubation period, the complexes formed were challenged with the addition of 1 μl of a mixture containing 100 μg/ml heparin, 600 nm UTP, and 600 nm CTP to allow RNA elongation. The reaction was stopped after 5 min with 5 μl of loading buffer, and the transcripts were separated by denaturing electrophoresis. The transcripts were detected by autoradiography using Kodak XAR film overnight at -20 °C, and promoter activity was quantified on a PhosphorImager SI (Molecular Dynamics; Amersham Biosciences) using ImageQuant 5.2 software. The percentage of templates transcribed was calculated by dividing the moles of 131-bp spoIIG transcript produced by the moles of template added to the reaction. The transcript produced was calculated from Cerenkov radiation in an excised gel slice containing the transcript, the number of G residues/transcript, and the specific activity of the [α32P]GTP in the reaction. The apparent van't Hoff enthalpy was calculated by taking advantage of the thermodynamic equivalence of the Gibb's free energy functions ΔG° = ΔH° - TΔS° = -RTlnKeq. Rearrangement yields the equation lnKeq = -(ΔH°/R)T-1 + ΔS°R-1, and plotting lnKeq as a function of T-1 yields a curve with a slope equivalent to -(ΔH°/R). The tangent to the resulting curve at Tm-1 permits the calculation of ΔH° (35Grimes E. Busby S. Minchin S. Nucleic Acids Res. 1991; 19: 6113-6118Crossref PubMed Scopus (18) Google Scholar, 36Haynie D.T. Biological Thermodynamics. Cambridge University Press, Cambridge2001: 98Google Scholar, 37Roe J.H. Record Jr., M.T. Biochemistry. 1985; 24: 4721-4726Crossref PubMed Scopus (64) Google Scholar, 38Saecker R.M. Tsodikov O.V. McQuade K.L. Schlax Jr., P.E. Capp M.W. Record Jr., M.T. J. Mol. Biol. 2002; 319: 649-671Crossref PubMed Scopus (92) Google Scholar). We have examined the influence of Spo0A∼P on the events underlying transcription initiation by using an extended series of templates that contain the nontemplate strand sequence on both strands over defined regions within the spoIIG promoter. In addition to those templates described earlier (29Rowe-Magnus D.A. Spiegelman G.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5305-5310Crossref PubMed Scopus (20) Google Scholar), the series included templates that were single-stranded at positions -14 to -9 inclusive relative to the transcription start site (NT14/9), -14 to -11 (NT14/11), or at positions -14 and -13 (NT14/13). These data were compared with those obtained using a pair of fully double-stranded promoters, the wild type spoIIG promoter, and a variant, IIG17, in which the spacer length has been reduced to a consensus 17 bp instead of the 22 bp observed in the wild type. The spoIIG promoter sequence and a diagram of the seven templates used in this study are shown in Fig. 1. Several transcripts were observed by denaturing polyacrylamide gel electrophoresis of the products from the in vitro transcription reactions using some of these templates. In the following analysis we discuss only the major transcript that had been shown to represent initiation at the position seen on wild type spoIIG in the presence of Spo0A∼P (29Rowe-Magnus D.A. Spiegelman G.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5305-5310Crossref PubMed Scopus (20) Google Scholar). Figs. 2A and 3A show autoradiograms of the section of the polyacrylamide gels containing the major transcript.Fig. 3Time course of initiated complex formation. A, autoradiographs of the primary products of in vitro transcription reactions. The templates are indicated at the left, and the times at which initiated complexes were challenged are indicated below each lane. Transcripts produced in the presence (left panel) and absence (right panel) of Spo0A∼P are shown. Transcription reactions were performed using spoIIG (B), IIG17 (C), NT14/13 (D), NT14/11 (E), NT14/9 (F), or NT14/3 (G) as the template. The levels of transcription indicative of the amount of initiated complex formed at spoIIG were plotted relative to transcription in the presence of Spo0A∼P after 120 s. The levels of transcription indicative of the amount of initiated complex formed at the other templates were plotted relative to transcription in the absence of Spo0A∼P after 120 s. The reactions containing either 800 nm Spo0A∼P (closed circles) or an equivalent buffer lacking Spo0A (open circles), 4 nm DNA template, and the initiating nucleotides ATP and GTP in 1× transcription buffer were prepared on ice. The reactions were incubated at 37 °C for 2 min, and then transcription was initiated with the addition of 1 μl (400 fmol) of RNAP. At the indicated times, samples were withdrawn and added to a 1-μl mixture containing heparin, UTP, and CTP. Elongation was allowed to proceed for 5 min before the reactions were terminated, and the transcription products separated by electrophoresis. Relative amounts of transcription were quantified as described under “Experimental Procedures.” The graphs represent averages from three independent experiments. The errors are less than 8% of the plotted values. The time required to achieve maximal formation of initiated complexes and the estimated effect of Spo0A∼P in stimulating the rate of this process are listed in Table I.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Levels of in Vitro Transcription from Heteroduplex Templates—RNAP alone directed only a low level of transcription from a template created by annealing fully wild type spoIIG DNA strands (Table I), whereas transcription was increased 6–50-fold for all the heteroduplex templates tested. In general, the amount of transcription from the predenatured templates in the absence of Spo0A∼P increased with the amount of single-stranded DNA. The exception, NT14/7, also gave rise to a transcript that utilized the opposite strand of the heteroduplex, which would be predicted to obstruct RNAP binding and reduce the amount of the appropriate transcript generated. The relative affinity of RNAP for the heteroduplex templates as determined by a gel mobility shift paralleled the relationship between the templates described above (data not shown). In addition, the fact that transcription initiation from the IIG17 promoter was independent of Spo0A∼P confirms the long held notion that Spo0A∼P is required to overcome the overlong spacer of the wild type promoter.Table ISummary of in vitro transcription dataTemplatePercent template transcribedaPercentage of template transcribed determined as described under “Experimental Procedures.” The errors represent the standard deviation from four experiments-Fold stimulationbAverages and standard deviations from 10 experimentsTmΔHapp,txncApparent van't Hoff enthalpy determined as described under “Experimental Procedures.” The errors are estimated to be <30% of the cited valueTime to maximal levels of initiated complexesRate stimulation by Spo0A≈PdAverages and standard deviation from three experiments-Spo0A≈P+Spo0A≈P°Ckcal mol-1sspoIIG0.6 ± 0.313 ± 218 ± 230426025 ± 6IIG1713 ± 212 ± 6≈1304260≈1NT14/134 ± 116 ± 63.5 ± 0.72833303.5 ± 0.2NT14/115 ± 115 ± 62.8 ± 0.82833304.9 ± 1.6NT14/918 ± 418 ± 5≈12424<10≈1NT14/711 ± 211 ± 4≈12424NDeND, not doneNDNT14/332 ± 830 ± 9≈12013<10≈1a Percentage of template transcribed determined as described under “Experimental Procedures.” The errors represent the standard deviation from four experimentsb Averages and standard deviations from 10 experimentsc Apparent van't Hoff enthalpy determined as described under “Experimental Procedures.” The errors are estimated to be <30% of the cited valued Averages and standard deviation from three experimentse ND, not done Open table in a new tab Effects of Spo0A∼P on in Vitro Transcription from Heteroduplex Templates—Because the influence of Spo0A is obscured by the variation in promoter strength, we calculated the fold stimulation of transcription by Spo0A∼P as the most meaningful comparison between the heteroduplexes. Spo0A∼P stimulated the level of transcription from the wild type spoIIG promoter ∼20-fold (Table I). By comparison, transcription from IIG17 and from the templates with the three largest predenatured regions, NT14/3, NT14/7, and NT14/9, was independent of Spo0A∼P. Spo0A∼P stimulated transcription from templates with the two smallest predenatured regions, NT14/11 and NT14/13, by 2.8- and 3.5-fold, respectively. This sharp transition to Spo0A dependence as the single-stranded region shrank from 6 to 4 bp (NT14/9 to NT14/11) indicated that the effect of Spo0A was limited to stages preceding the complete exposure of the nontemplate strand of the -10 element. We tested a variety of other templates. Templates containing mismatches at -28 and -27 supported only low levels of transcription by RNAP alone and were indistinguishable from the wild type promoter in their properties (data not shown). This suggested that the single-stranded regions do not simply increase template flexibility that might help to align the -35 and -10 sequences on the spoIIG promoter with the appropriate regions in the σ subunit. Furthermore, heteroduplexes containing the template strand sequence on both strands exhibited reduced levels of transcription that correlated with the extent of nontemplate strand -10 element consensus sequence retained (data not shown). These results are consistent with the interpretation that interactions between σA and single-stranded DNA from the nontemplate strand of the -10 element were essential for holoenzyme-directed transcription. Temperature Dependence of Transcription from Heteroduplex Templates—Because Spo0A∼P is required to form an open complex at spoIIG (29Rowe-Magnus D.A. Spiegelman G.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5305-5310Crossref PubMed Scopus (20) Google Scholar) and because separation of the DNA strands is widely believed to be an energy-intensive process (39Helmann J.D. deHaseth P.L. Biochemistry. 1999; 38: 5959-5967Crossref PubMed Scopus (132) Google Scholar, 40deHaseth P.L. Helmann J.D. Mol. Microbiol. 1995; 16: 817-824Crossref PubMed Scopus (128) Google Scholar), we reasoned that Spo0A might facilitate open complex formation by reducing an energetic barrier to transcription. It seemed possible then that lower reaction temperatures might enhance the dependence of transcription on Spo0A∼P even on templates with denatured regions. Denaturing additional base pairs could also require additional energetic input so that transcription from templates containing smaller predenatured regions might be more sensitive to reduced temperature. Consequently, we investigated the effect of Spo0A∼P on the temperature dependence of transcription from each of these templates. Without Spo0A∼P transcription from the fully duplexed wild type spoIIG promoter was low at all temperatures, although a slight increase in transcription with increasing temperature could be detected (Fig. 2B). Even in the presence of Spo0A∼P, below 22 °C the level of transcription from the wild type spoIIG promoter was less than 4% of that observed at 37 °C. The amount of transcription increased dramatically between 27 and 37 °C, and the transition temperature (Tm) at which point the transcription was half-maximal was ∼30 °C (Table I). This value was higher than expected from studies with E. coli RNAP where the Tm is typically 25 °C under similar reaction conditions (41Nakanishi S. Adhya S. Gottesman M. Pastan I. J. Biol. Chem. 1975; 250: 8202-8208Abstract Full Text PDF PubMed Google Scholar). We had anticipated that reducing the overlong spacing at the spoIIG promoter would both obviate the requirement for Spo0A∼P and, because of the appropriate phasing of the conserved promoter elements, would be less energetically demanding. Although transcription from IIG17 was independent of Spo0A (Fig. 2C), the thermal profile of transcription from IIG17 was indistinguishable from spoIIG, both in terms of its" @default.
• W2137767952 created "2016-06-24" @default.
• W2137767952 creator A5080504416 @default.
• W2137767952 creator A5085051527 @default.
• W2137767952 date "2004-04-01" @default.
• W2137767952 modified "2023-10-01" @default.
• W2137767952 title "The Bacillus subtilis Response Regulator Spo0A Stimulates σA-Dependent Transcription Prior to the Major Energetic Barrier" @default.
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