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• W2008274524 abstract "Twenty-one conserved positively charged and aromatic amino acids between residues 331 and 462 of sigma 54 were changed to alanine, and the mutant proteins were studied by transcription, band shift analysis, and footprinting in vitro. A small segment corresponding to the rpoN box was found to be most important for binding duplex DNA. Two amino acids, 52 residues apart, were found to be critical for maintaining transcriptional silencing in the absence of activator. These two activator bypass mutants and several other mutants failed to bind the type of fork junction DNA thought to be required to maintain silencing. The two bypass mutants showed a binding pattern to DNA probes that was unique, both in comparison to other C-terminal mutants and to previously known N-terminal bypass mutants. On this basis, a model is proposed for the role of the C terminus and the N terminus of sigma 54 in enhancer-dependent transcription. Twenty-one conserved positively charged and aromatic amino acids between residues 331 and 462 of sigma 54 were changed to alanine, and the mutant proteins were studied by transcription, band shift analysis, and footprinting in vitro. A small segment corresponding to the rpoN box was found to be most important for binding duplex DNA. Two amino acids, 52 residues apart, were found to be critical for maintaining transcriptional silencing in the absence of activator. These two activator bypass mutants and several other mutants failed to bind the type of fork junction DNA thought to be required to maintain silencing. The two bypass mutants showed a binding pattern to DNA probes that was unique, both in comparison to other C-terminal mutants and to previously known N-terminal bypass mutants. On this basis, a model is proposed for the role of the C terminus and the N terminus of sigma 54 in enhancer-dependent transcription. nitrogen regulator protein C nitrogen fixation H Sigma 54 is unique among bacterial sigma factors with regard to both amino acid sequence and transcription mechanism. It is not a member of the sigma 70 family of proteins and is uniquely required to transcribe from enhancer-dependent promoters. As with most sigmas, promoter recognition involves two DNA sequence elements separated by a defined number of base pairs. Initially the holoenzyme binds the two sigma 54-specific promoter elements, termed −24 and −12 in reference to locations that include conserved nucleotides (1Merrick M.J. Mol. Microbiol. 1993; 10: 903-909Crossref PubMed Scopus (338) Google Scholar). The holoenzyme typically remains tightly bound until signal transduction leads to activation by a protein bound to a remote enhancer element (2Reitzer L.J. Magasanik B. Cell. 1986; 45: 785-792Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 3Morett E. Buck M. J. Mol. Biol. 1989; 210: 65-77Crossref PubMed Scopus (180) Google Scholar, 4Sasse-Dwight S. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8934-8938Crossref PubMed Scopus (190) Google Scholar, 5Popham D.L. Szeto D. Keener J. Kustu S. Science. 1989; 243: 629-635Crossref PubMed Scopus (314) Google Scholar). The holoenzyme is initially inactive, because it cannot open the DNA and engage the transcription start site; the enhancer protein overcomes this block, thus allowing the DNA to open and transcription to begin (reviewed in Ref. 6Gralla J.D. Nature Struct. Biol. 2000; 7: 530-532Crossref PubMed Scopus (9) Google Scholar). The use of enhancers and the common regulation at the DNA melting step differentiates this class of holoenzymes from all others in bacteria (reviewed in Ref. 7Buck M. Gallegos M. Studholme D. Guo Y. Gralla J. J. Bacteriol. 2000; 182: 4129-4136Crossref PubMed Scopus (349) Google Scholar). All sigma factors use the common core enzyme so these differences are solely attributed to the nature of the sigma factor. In addition to binding duplex DNA, sigma factors bind single-stranded DNA and structures with single strand and duplex DNA juxtaposed (fork junction structures, Ref. 8Guo Y. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11655-11660Crossref PubMed Scopus (87) Google Scholar). In the case of sigma 54, these latter interactions are central to control by the holoenzyme (6Gralla J.D. Nature Struct. Biol. 2000; 7: 530-532Crossref PubMed Scopus (9) Google Scholar, 9Guo Y. Wang L. Gralla J.D. EMBO J. 1999; 18: 3746-3756Crossref PubMed Scopus (158) Google Scholar, 10Guo Y. Lew C. Gralla J. Genes Dev. 2000; 14: 2242-2255Crossref PubMed Scopus (63) Google Scholar, 11Cannon W.V. Gallegos M.-T. Buck M. Nature Struct. Biol. 2000; 7: 594-601Crossref PubMed Scopus (92) Google Scholar). Within the inactive closed complex containing sigma 54 holoenzyme, a single base pair adjacent to the −12 recognition element is transiently melted (12Morris L. Cannon W. Claverie-Martin F. Austin S. Buck M. J. Biol. Chem. 1994; 269: 11563-11571Abstract Full Text PDF PubMed Google Scholar). This provides a transient double-strand/ single-strand fork junction structure. Interaction at this fork is repressive in the sense that the conformation of sigma bound to it helps keep the holoenzyme silent by blocking its ability to melt DNA. Activators can overcome this silencing by triggering conformational changes in both sigma and holoenzyme (6Gralla J.D. Nature Struct. Biol. 2000; 7: 530-532Crossref PubMed Scopus (9) Google Scholar, 10Guo Y. Lew C. Gralla J. Genes Dev. 2000; 14: 2242-2255Crossref PubMed Scopus (63) Google Scholar, 11Cannon W.V. Gallegos M.-T. Buck M. Nature Struct. Biol. 2000; 7: 594-601Crossref PubMed Scopus (92) Google Scholar). Both the silent state and the active state rely on a complex network of interactions that centrally involve the promoter −12 element (9Guo Y. Wang L. Gralla J.D. EMBO J. 1999; 18: 3746-3756Crossref PubMed Scopus (158) Google Scholar, 13Wang J.T. Syed A. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9538-9543Crossref PubMed Scopus (53) Google Scholar, 14Wang L. Gralla J.D. J Bacteriol. 1998; 180: 5626-5631Crossref PubMed Google Scholar, 15Wang L. Guo Y. Gralla J.D. J Bacteriol. 1999; 181: 7558-7565Crossref PubMed Google Scholar). Interactions with the −24 elements are simpler and are the dominant factor in directing general DNA binding (16Sasse-Dwight S. Gralla J.D. Cell. 1990; 62: 945-954Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 17Hsieh M. Gralla J.D. J. Mol. Biol. 1994; 239: 15-24Crossref PubMed Scopus (35) Google Scholar, 18Wong C. Tintut Y. Gralla J.D. J. Mol. Biol. 1994; 236: 81-90Crossref PubMed Scopus (56) Google Scholar). The motifs on sigma 54 that direct these various DNA interactions are not well established but appear to involve primarily N-terminal and C-terminal sequences. A short N-terminal region is required for regulation; numerous deregulated bypass forms of sigma have been identified with changes in the first 50 amino acids (19Wang J.T. Syed A. Hsieh M. Gralla J.D. Science. 1995; 270: 992-994Crossref PubMed Scopus (84) Google Scholar, 20Syed A. Gralla J.D. Mol. Microbiol. 1997; 23: 987-995Crossref PubMed Scopus (25) Google Scholar, 21Kelly M.T. Hoover T.R. J. Bacteriol. 1999; 181: 3351-3357Crossref PubMed Google Scholar, 22Casaz P. Gallegos M.T. Buck M. J. Mol. Biol. 1999; 292: 229-239Crossref PubMed Scopus (30) Google Scholar). Holoenzymes containing these mutant forms of sigma can transcribein vitro in the absence of activator. These holoenzymes have also lost the capacity to bind the fork junction structure associated with the silent state of the closed complex. They have also gained an ability to interact with downstream single-stranded DNA (9Guo Y. Wang L. Gralla J.D. EMBO J. 1999; 18: 3746-3756Crossref PubMed Scopus (158) Google Scholar). By contrast, mutations in the C-terminal region can destroy general DNA binding and this property is not shared by N-terminal mutants (16Sasse-Dwight S. Gralla J.D. Cell. 1990; 62: 945-954Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 18Wong C. Tintut Y. Gralla J.D. J. Mol. Biol. 1994; 236: 81-90Crossref PubMed Scopus (56) Google Scholar). Because the −24 interaction is dominant for DNA binding, it is presumed that the C terminus recognizes this element. The C-terminal region also contains determinants that are needed for activation of the holoenzyme (21Kelly M.T. Hoover T.R. J. Bacteriol. 1999; 181: 3351-3357Crossref PubMed Google Scholar, 23Chaney M. Buck M. Mol. Microbiol. 1999; 33: 1200-1209Crossref PubMed Scopus (48) Google Scholar) and both ends of the protein have been proposed to participate in recognition of the −12 promoter element (17Hsieh M. Gralla J.D. J. Mol. Biol. 1994; 239: 15-24Crossref PubMed Scopus (35) Google Scholar, 24Merrick M. Chambers S. J. Bacteriol. 1992; 174: 7221-7226Crossref PubMed Google Scholar). Recently a deregulated, bypass point mutation has been identified within the C-terminal region (23Chaney M. Buck M. Mol. Microbiol. 1999; 33: 1200-1209Crossref PubMed Scopus (48) Google Scholar). It is obvious that the C-terminal region of sigma 54 has a particularly complex array of functions. Subregions of potential importance within the C terminus have been identified. Among these are the following: a block of 10 almost completely conserved amino acids termed the rpoN box (25van Slooten J.C. Broughton W.J. Wong C.H. Stanley J. J. Bacteriol. 1990; 172: 5563-5574Crossref PubMed Google Scholar), a segment originally suggested to have the potential to form a helix-turn-helix (HTH, Ref. 26Coppard J.R. Merrick M.J. Mol. Microbiol. 1991; 5: 1309-1317Crossref PubMed Scopus (36) Google Scholar), and a segment that can be cross-linked to DNA (27Cannon W. Claverie-Martin F. Austin S. Buck M. Mol. Microbiol. 1994; 11: 227-236Crossref PubMed Scopus (40) Google Scholar). Collectively, these segments and others that have proposed functions (28Guo Y. Gralla J.D. J. Bacteriol. 1997; 179: 1239-1245Crossref PubMed Google Scholar) cover a region of ∼150 amino acids. This extensive region may contain activities that contribute to recognition of duplex DNA, fork junction DNA, and single strand DNA. In studies of sigma 54 and sigma 70, the most prominent residues involved in DNA recognition are positively charged and aromatic amino acids (24Merrick M. Chambers S. J. Bacteriol. 1992; 174: 7221-7226Crossref PubMed Google Scholar, 28Guo Y. Gralla J.D. J. Bacteriol. 1997; 179: 1239-1245Crossref PubMed Google Scholar, 29Fenton M. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Crossref PubMed Scopus (110) Google Scholar, 30Oguiza J.A. Gallegos M.-T. Chaney M.K. Cannon W.V. Buck M. Mol. Microbiol. 1999; 33: 873-885Crossref PubMed Scopus (15) Google Scholar, 31Taylor M. Butler R. Chambers S. Casimiro M. Badii F. Merrick M. Mol. Microbiol. 1996; 22: 1045-1054Crossref PubMed Scopus (44) Google Scholar, 32Helmann J.D. deHaseth P.L. Biochemistry. 1999; 38: 5959-5967Crossref PubMed Scopus (132) Google Scholar). The C terminus of sigma 54 contains many such residues, and these have a tendency to cluster within the motifs suggested to be important. To learn the role of these residues and to identify the array of functions within the C terminus we have mutated each of these residues individually, purified the mutant proteins, and characterized them using biochemical assays. The collection displays a rich array of properties, which allows the role of the C terminus in both regulation and DNA binding to be understood to a much greater extent. The plasmid pAS54, derived from expression plasmid pJF5401, carries theEscherichia coli sigma 54 gene (33Syed A. Gralla J.D. J. Bacteriol. 1998; 180: 5619-5625Crossref PubMed Google Scholar). This plasmid was subjected to site-directed mutagenesis at the desired positions with QuikChange site-directed mutagenesis kit (Stratagene). The presence of the correct mutation was confirmed by sequencing. The strain E. coli YMC109 lacking a wild-type chromosomal copy of the sigma 54 gene was used as the host. Sigma 54 and its derivatives were partially purified by modified methods based on those described (13Wang J.T. Syed A. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9538-9543Crossref PubMed Scopus (53) Google Scholar). In brief, 10 ml of Luria-Bertani medium with 100 μg/ml ampicillin was inoculated with YMC109 cells transformed with expression vector that carries the appropriate sigma 54 mutant. The cell culture was grown to 1 OD at 30 °C with vigorous aeration. The culture was shifted to 43 °C and grown for another 3–4 h to induce expression. Cells were collected and suspended in 300 μl of buffer S (10 mmTris-HCl, pH 8.0, 200 mm KCl, 0.1 mm EDTA, 1 mm dithiothreitol, and 5% glycerol) and disrupted by sonication. After centrifugation of the cell lysate, the pellet containing the inclusion body was dissolved in 150 μl of buffer S plus 4 m guanidine-HCl and 0.1% Nonidet P-40 (nonionic detergent). Sonication was used to help dissolve the pellet, which was then dialyzed, first against buffer S with 1 mguanidium-HCl, then against buffer S, and finally against buffer S with 40% glycerol. After each dialysis, the undissolved material was discarded. The protein concentration was estimated on an SDS-polyacrylamide gel against known protein markers. Standard one-round in vitro transcription was used as described previously (13Wang J.T. Syed A. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9538-9543Crossref PubMed Scopus (53) Google Scholar). The activated transcription reaction mixture contained 100 nm NtrC,1 45 nm sigma 54 holoenzyme (core RNA polymerase is from Epicentre Technology), 5 nm supercoiled DNA template pTH8, 10 mm carbamyl phosphate, 0.05 mm GTP, 0.05 mm CTP, 4 μCi of [32P]UTP, 50 μm unlabeled UTP, and 3 mm ATP in transcription buffer (50 mm HEPES, pH 7.8, 50 mm KCl, 10 mm MgCl2, 0.1 mm EDTA, 1 mm dithiothreitol, 50 ng of bovine serum albumin, and 3.5% polyethylene glycol), in a total reaction volume of 10 μl. When NtrC is present, the reaction mixture was preincubated without GTP, UTP, and CTP for 20 min at 37 °C. When NtrC is absent, the reaction mixture is preincubated with GTP and CTP for 20 min at 37 °C. ATP was always present at the concentration of 3 mm. After preincubation, the missing nucleotides and heparin (final concentration, 100 μg/ml) were added to the reaction mixture and incubated for 10 min. The reaction was stopped by the addition of urea-saturated formamide dye, and the mixtures were loaded on 6% denaturing polyacrylamide gels for electrophoresis. The data were analyzed with a phosphorimager. The band shift analysis was done as described (9Guo Y. Wang L. Gralla J.D. EMBO J. 1999; 18: 3746-3756Crossref PubMed Scopus (158) Google Scholar). Briefly, the probes were prepared by annealing two complementary DNA strands. The lengths of bottom strand and top strand are specifically described in the legends. The annealing mixture contained 4 pmol of labeled bottom-strand DNA and 6 pmol of top strand in 10 mm HEPES, pH 7.9, 80 mm NaCl. The mixture was boiled for 2 min and gradually cooled to room temperature. Annealing was monitored by 10% polyacrylamide gel electrophoresis. 10 μl of band shift assay mixture contained 1 nm DNA, 15 nm RNA polymerase, and 6.0 ng of poly(dI-dC) per μl in 1× HEPES buffer (50 mm HEPES-HCl, pH 7.9, 100 mm KCl, 10 mm MgCl2, 0.1 mm EDTA, 1 mm dithiothreitol, 0.05 μg/ml bovine serum albumin, 2.8% polyethylene glycol 8000). To detect the induced interaction by NtrC, 625 nm NtrC was added into the mixture to act from solution. The mixtures were incubated at 37 or 15 °C for 10 min and subjected to 5% polyacrylamide gel electrophoresis, which was run at 350 V in ice. After electrophoresis, the radioactive bands were visualized and analyzed with a phosphorimager. The nifH promoter fragment from −59 to +29 was synthesized by Operon technology. The bottom strand DNA was labeled with [γ-32P]ATP and annealed with the complementary top strand. The 10-μl reaction mixture contained 0.8 nm DNA, 45 nm holoenzyme, and 6.0 ng of dI-dC per μl in 1× HEPES buffer (as described above) and incubated at 37 °C for 10 min. Then 0.8 μl of DNase I solution (1 mg/ml in 10 mm MgCl2 and 5 mmCaCl2) was added into the mixture and incubated for 1 min. The reaction was stopped with 10 μl of stop solution (20 mm EDTA, pH 8, 1% (w/v) SDS, 0.2 m NaCl). The DNA was precipitated and dissolved in 5 μl of urea-saturated formamide dye and loaded on 10% denaturing polyacrylamide gels for electrophoresis. The data were analyzed with a phosphorimager. We identified 21 highly conserved positively charged and aromatic amino acids within the 150 amino acid region from residue 319 to 469. Each of these was conserved in at least 18 of the 21 sigma 54 sequences available at the start of this study. They were changed individually to alanine by site-directed mutagenesis. The plasmids carrying mutated sigma 54 genes were transferred individually to a strain lacking endogenous sigma 54. The 21 mutants and the wild-type sigma protein were partially purified in 2 batches of 11 proteins each. In general the proteins were judged to be 50–80% pure. The single exception was R383A, which was poorly induced, possibly because of a folding defect, and thus was much less pure. As a comparison for this form of sigma a mock purification was done from a strain lacking plasmid and thus containing only contaminating proteins. The 22 forms of sigma were used in equal amounts for biochemical experiments, as judged from staining of the protein gels. The holoenzymes were formed using a 4:1 ratio of sigma to core. Although the N terminus is the primary locus of in vitro deregulated bypass mutants, a recent in vitro study found this property to be associated with a C-terminal change (23Chaney M. Buck M. Mol. Microbiol. 1999; 33: 1200-1209Crossref PubMed Scopus (48) Google Scholar). This mutant, R336A, is within the collection just described. To determine whether R336 is unique or belongs to a family that determines the requirement for activator, all altered sigma 54 holoenzymes were tested with a protocol for activator-independent transcription in vitro. The activator-independent bypass transcription assay was done using plasmid pTH8 that carries the glnAP2 promoter as a template (19Wang J.T. Syed A. Hsieh M. Gralla J.D. Science. 1995; 270: 992-994Crossref PubMed Scopus (84) Google Scholar). Holoenzyme, template DNA and ATP, GTP, and CTP were preincubated to allow the potential for initiation without activator. Then, heparin was added to destroy any residual closed complex. [32P]UTP was added to initiate a round of transcription. No activator was included, and in control experiments one round of activated transcription with wild-type holoenzyme was used for comparison. The template contains a downstream terminator, permitting a discrete RNA to be assayed. In this assay, wild-type and 19 of the 21 mutant proteins showed very low levels of activator-independent transcription, less than 5% of the amount produced by activated wild-type holoenzyme (Fig.1). Two of the mutant sigmas give high transcription levels. R336A gives a 90% signal in this assay, consistent with expectations from the prior report (23Chaney M. Buck M. Mol. Microbiol. 1999; 33: 1200-1209Crossref PubMed Scopus (48) Google Scholar). In addition K388A gives a high signal, at approximately the 50% level. We note that these residues are not close to each other. One is within the region that can cross-link to DNA, and the other is within or directly adjacent to the putative HTH motif. We also note that residues that are very close to each of these bypass mutants, such as Lys-331 and Arg-342 that surround Arg-336, and Arg-383, Tyr-389, and Arg-394 that surround Lys-388, do not show this property when mutated. These properties and others to be assayed below are collected in TableI. We infer that silencing in the absence of activator is conferred by widely separated amino acids within the C terminus. In contrast to bypass mutants in the N terminus, the silencing determinants do not involve extensive adjacent stretches of amino acids.Table IMutant propertiesSigma 54bypass trxn1-atrxn, transcription.Activated trxnDNA bindingT12 bindingT9 bindingsupershift37 °C15 °CDNaseIgel shiftWT−+++++++++++++++++K331A−+++++/++++++++R336A+++++++−+++/++R342A−+++++++++++++F355A−+++/−+++/−+K363A−++++++++++++++R383A−+/−+/−−+/−+/−+/−+/−K388A++++/++++/−−++Y389A−+++++++++++++++++R394A−+++++++++++++/++++F397A−+++++++++++++++++K400A−+++++/−−−+Y401A−++++++++++++/−++R402A−+++++++++++++++F403A−+/+++/−+/−+/−−−+/−R421A−+++++−−+K425A−+++/+++/+++/+++/−+/−+/++R455A−+/−−+/−+/−−−−R456A−−−−−−−−K460A−+++++−−+Y461A−+/−−−−−−−R462A−−−+/−−−−−1-a trxn, transcription. Open table in a new tab Next, the 22 proteins were assayed for transcription using a protocol that strictly depends on activator. In this protocol heparin is added after activator but prior to addition of initiating nucleotides. The experiment is done under optimal conditions for in vitro transcription, including 37 °C and high concentrations of all proteins. Under these conditions transcription depends strictly on activator with very little bypass transcription. This stringency was shown in prior experiments using strong N-terminal bypass mutants (19Wang J.T. Syed A. Hsieh M. Gralla J.D. Science. 1995; 270: 992-994Crossref PubMed Scopus (84) Google Scholar) and is also true for the C-terminal bypass mutants just described (not shown). Fig.2 shows the results of this activated transcription assay using the mutant holoenzymes. The data from several experiments is compiled in Fig. 2 B, where the transcription level is normalized to that of the wild-type holoenzyme. Under these conditions, 15 of the 21 mutant holoenzymes behave normally, giving a level of activated transcription not significantly different from wild-type. Of the six defective mutants, four are in the rpoN box region, K455A, R456A, Y461A, and R462A. A fifth mutant is R383A, which because of its low purity cannot be firmly assigned as being defective in activated transcription. The remaining mutant, F355A is the least defective of the group (see also Ref. 30Oguiza J.A. Gallegos M.-T. Chaney M.K. Cannon W.V. Buck M. Mol. Microbiol. 1999; 33: 873-885Crossref PubMed Scopus (15) Google Scholar). We infer that residues within the rpoN box region are particularly important for obtaining a signal in the activated transcription experiment. Other segments of the C terminus seem to be less important in this assay. We note that the two mutants that gave bypass transcription in Fig. 1 are not in the rpoN box, suggesting that the determinants for silencing and those required to lead to activated transcription are at least partially separable. In the next experiment, the efficiency of the activated transcription assay was lowered to see if secondary defects would turn up in residues outside the rpoN box region. This simply involved lowering the temperature from 37 to 15 °C. Under these conditions, several other mutants showed partial defects (Fig. 3). Now only 8 of the 21 mutants retained a transcription level of greater than 80% of the wild-type protein (Fig. 3 B). The four mutants in the rpoN box that showed defects under optimal conditions were now even more defective. R383A and F355A were not much affected by the change in temperature. Seven mutants showed defects only in this assay, and they were not clustered in any region, being scattered between amino acids 336 and 460. It appears that residues throughout the C-terminal region play some role in activated transcription. These include Arg-336 and Lys-388, which play a primary role in silencing and now are seen to have their activated transcription levels reduced to half of the wild-type level under these suboptimal conditions. We note that the data makes no clear distinction between positively charged and aromatic residues; both types are among the six residues that are most defective and among the eight residues that show no defect in either assay. These data indicate that the rpoN box residues play a primary role in transcription. A secondary role is played by residues scattered throughout the C-terminal 150 amino acids. As closed complex formation is a prerequisite for transcription we next assayed duplex DNA binding using the 21 mutant holoenzymes. The stability of closed complexes is generally less than that of open complexes and so two different assays were used, DNase footprinting and a band shift analysis. A fragment of the nifH promoter, which contains the central consensus sequence of the −24 and −12 elements, was used in both assays. Closed complex footprints are known to cover these two elements but yield incomplete protection; the interaction does not extend very far beyond the elements, in particular leaving the transcription start site unprotected (34Cannon W. Claverie-Martin F. Austin S. Buck M. Mol. Microbiol. 1993; 8: 287-298Crossref PubMed Scopus (46) Google Scholar, 35Cannon W. Missailidis S. Smith C. Cottier A. Austin S. Moore M. Buck M. J. Mol. Biol. 1995; 248: 781-803Crossref PubMed Scopus (62) Google Scholar). Under the conditions of our experiment, the overall promoter occupancy with wild-type holoenzyme in closed complexes was 50–70% for the best protected positions in optimal experiments. As seen in Fig. 4, the footprints gave partial protection over the −12 and −24 elements (compare the two leftmost lanes in Fig. 4 A) and no protection further downstream. We used three of the strongest bands to analyze the quantitative extent of protection using phosphorimager technology. Fig. 4 A shows that position −1 is of equal intensity in the two leftmost lanes, without and with wild-type holoenzyme. This band is used to normalize the signal to ensure that binding by the 21 mutants is not improperly analyzed because of altered loading or extent of digestion. The degree of protection was judged by comparing the signals of mutants with wild type at the −21 and −10 band positions. That is, after normalizing the signal from each lane to equalize the −1 band intensity, the protection using bands at −21 or −10 was taken as the extent of binding in the closed complex. The data were not significantly different using the two bands, and the results using −10 are quantified in Fig. 4 B. In this display, wild type is taken as 1.0 so the extent of protection by each mutant can be compared directly to wild type. The most dramatic result is that each of four mutations in the rpoN box lead to by far the lowest extent of protection (R455A, R456A, Y461A, and R462A in Fig. 4 B). R455A and R456A have been shown elsewhere to bind core polymerase and form holoenzymes, so this is not the source of the defect in DNA binding (31Taylor M. Butler R. Chambers S. Casimiro M. Badii F. Merrick M. Mol. Microbiol. 1996; 22: 1045-1054Crossref PubMed Scopus (44) Google Scholar). These four mutants are the same ones that showed the greatest defect in transcription so the cause of this is very likely to be an inability to bind duplex DNA to form a closed complex. The fifth change in the rpoN box, K460A, is partially down in DNA binding, mimicking its partial loss of transcription under suboptimal conditions (compare Figs. 4 Band 3 B). Other mutations, scattered about the C-terminal region, show partial reductions in closed complex formation using this assay. These partially defective mutants range over a nearly 150-amino acid region. This is roughly the same collection of mutants that showed partial defects in transcription under suboptimal conditions. Moreover, approximately one-third of the mutants show little evidence of defects, being at or near the levels of wild-type holoenzyme in both this assay and the suboptimal transcription assay. It appears that both binding and transcription have similar amino acid requirements. Because protection within closed complexes is fairly weak, we sought to confirm these results using a band shift assay. At very high concentrations of holoenzyme most mutants bound DNA well (not shown), and so the concentration was lowered to 15 nm to reveal significant differences (Fig. 5). Two shifted bands could be seen, a lower one that was not very sensitive to protein concentration or to mutation and an upper one with an intensity that varied with the protein type and its concentration (Fig.5 A). The upper band was used in the quantitative analysis (see Fig. 5 B). For the most part, the result of the band shift analysis was consistent with that of the footprint analysis. There were only three exceptions; F355A, F402A, and R383A, which bound better in the band shift analysis than in the footprint analysis. These three mutants were partially defective in both assays, and the higher extent of binding seen in band shifts was better correlated with the extent of transcription under suboptimal conditions. To learn if the level of binding by R383A was specific, we compared it to a mock-purified protein preparation obtained from nontransformed cells (Fig. 5 A,host). No upper band was present in the mock control, indicating that the R383A signal likely comes from the cloned sigma 54. However, because of the low purity of this protein and the uncertainty that misfolding is the cause of its low induction, we cannot be certain of the cause of the lowering of its binding to DNA. These experiments were repeated at lower temperature (15 °C, not shown), but the results were not different. Four of five rpoN box mutants showed very little binding in both footprint and band shift assays. Several other mutants (K388A, K400A, F403A, R421A, and K460A), showed reduced binding in both assays. It appears that the rpoN box region is the most critical for DNA binding, but other residues throughout the C terminus make an important quantitative contribution to affinity. In many cases, the lowering of occupancy within closed complexes likely accounts for the lowering of activated transcription under suboptimal conditions. After init" @default.
• W2008274524 created "2016-06-24" @default.
• W2008274524 creator A5059596664 @default.
• W2008274524 creator A5073216396 @default.
• W2008274524 date "2001-03-01" @default.
• W2008274524 modified "2023-09-29" @default.
• W2008274524 title "Roles for the C-terminal Region of Sigma 54 in Transcriptional Silencing and DNA Binding" @default.
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