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• W2061430251 abstract "ς38 is a non-essential but highly homologous member of the ς70 family of transcription factors. In vitro mutagenesis and in vivo screening were used to identify 22 critical amino acids in the promoter interaction domain of Escherichia coliς38. Electrophoretic mobility shift assay studies showed that residues involved in duplex DNA binding largely segregated into distinct regions that coincided with those of ς70. However, the majority of these amino acids were in non-conserved positions. Analysis indicates that this region of the two ςs probably has a common overall organization but differs in how its amino acids are used to form functional open complexes. Placement of the mutations on the known ς70 holoenzyme structure shows two clusters; one appears to be used for duplex DNA recognition and the other for the subsequent isomerization events. Permanganate assays for DNA melting support this view. ς38 is a non-essential but highly homologous member of the ς70 family of transcription factors. In vitro mutagenesis and in vivo screening were used to identify 22 critical amino acids in the promoter interaction domain of Escherichia coliς38. Electrophoretic mobility shift assay studies showed that residues involved in duplex DNA binding largely segregated into distinct regions that coincided with those of ς70. However, the majority of these amino acids were in non-conserved positions. Analysis indicates that this region of the two ςs probably has a common overall organization but differs in how its amino acids are used to form functional open complexes. Placement of the mutations on the known ς70 holoenzyme structure shows two clusters; one appears to be used for duplex DNA recognition and the other for the subsequent isomerization events. Permanganate assays for DNA melting support this view. The alternative ς factor, ς38 (rpoS), is the principle regulator of the general stress response inEscherichia coli. The ς38 regulon controls 50 to 100 genes (1Schellhorn H.E. Audia J.P. Wei L.I. Chang L. J. Bacteriol. 1998; 180: 6283-6291Google Scholar). Subsets of these genes are induced during starvation for various nutrients and in response to various stresses such as the accumulation of reactive oxygen species, changes in pH, and osmolarity. The highest activity of ς38 occurs during stationary phase when these and other stresses are present. Many factors contribute to this activity, especially the increased stability of the ς38 protein (2Hengge-Aronis R. Curr. Opin. Microbiol. 1999; 2: 148-152Google Scholar).ς38 is highly homologous to ς70, the vegetative ς factor responsible for the transcription of most of the housekeeping genes. The regions of ς70 that recognize promoter DNA, conserved region 4.2 of the protein (recognizes the −35 element), and conserved regions 2.3–2.5 (−10 element) are over 70% similar (60% identical) to those of ς38 (3Lonetto M. Gribskov M. Gross C.A. J. Bacteriol. 1992; 174: 3843-3849Google Scholar). However, ς38 promoters generally contain only a single DNA recognition element, a −10 sequence centered between nucleotides −14 and −7 (4Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Google Scholar, 5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar). The four most conserved of these nucleotides, −13C, −12T, −11A, and −7T, are involved in directing promoter selectivity and play a dominant role in setting promoter strength (4Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Google Scholar, 5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar, 6Gaal T. Ross W. Estrem S.T. Nguyen L.H. Burgess R.R. Gourse R.L. Mol. Microbiol. 2001; 42: 939-954Google Scholar). Three of these positions, −12T, −11A, and −7T, are also critical for ς70 function, although they are utilized at different steps during ς70 transcription initiation (7Fenton M.S. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9020-9025Google Scholar).ς38 and ς70 do not respond to regulators and the physiological state of the cell in the same manner. It is clear that such regulators as Lrp, CRP, H-NS, and many others can differentially effect transcription by ς38 and ς70 (8Bouvier J. Gordia S. Kampmann G. Lange R. Hengge-Aronis R. Gutierrez C. Mol. Microbiol. 1998; 28: 971-980Google Scholar, 9Colland F. Barth M. Hengge-Aronis R. Kolb A. EMBO J. 2000; 19: 3028-3037Google Scholar). At certain promoters, a low supercoiled state of the DNA, which is present in stationary phase (10Balke V.L. Gralla J.D. J. Bacteriol. 1987; 169: 4499-4506Google Scholar), seems to favor ς38 transcription (11Kusano S. Ding Q. Fujita N. Ishihama A. J. Biol. Chem. 1996; 271: 1998-2004Google Scholar). Increased concentrations of trehalose (12Kusano S. Ishihama A. J. Bacteriol. 1997; 179: 3649-3654Google Scholar), as well as potassium glutamate (13Ding Q. Kusano S. Villarejo M. Ishihama A. Mol. Microbiol. 1995; 16: 649-656Google Scholar), also preferentially stimulate ς38-dependent transcription at certain promoters.The basis for these diverse properties between the two highly homologous holoenzymes have remained largely unknown. Some differences in ς38 function have been attributed to differential recognition of nucleotides −14 and −13 (5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar) and a C-terminal “tail” that helps in sensing potassium glutamate (14Ohnuma M. Fujita N. Ishihama A. Tanaka K. Takahashi H. J. Bacteriol. 2000; 182: 4628-4631Google Scholar). The ς38 amino acids responsible for use of nucleotides −12 to −7 have not been characterized. Presumably, they would lie in the same part of ς70 that recognizes the −10 element, conserved regions 2.3–2.5. Because ς38 and ς70 promoters behave differently their interactions with region 2 may not be identical. There have been several studies identifying ς70 amino acids that use the −10 element (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar, 16Tomsic M. Tsujikawa L. Panaghie G. Wang Y. Azok J. deHaseth P.L. J. Biol. Chem. 2001; 276: 31891-31896Google Scholar), but information concerning ς38 recognition is sparse.One purpose of this study is to identify functionally important residues in the homologous region 2 of ς38. Another purpose is to begin to understand how these amino acids function. The results will be interpreted using the known structure of the DNA interaction region of ς70, and models for promoter usage by both ςs will be discussed.RESULTSRegions 2.3, 2.4, and 2.5 of ς70 have been identified as the critical components that recognize and melt the −10 element (21Daniels D. Zuber P. Losick R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8075-8079Google Scholar, 22deHaseth P.L. Helmann J.D. Mol. Microbiol. 1995; 16: 817-824Google Scholar). To understand their role in ς38(rpoS), this DNA segment was mutated by error-prone PCR and then ligated into a receiver rpoS vector. The mutant plasmid collection was transformed into an rpoS minus strain that contained a lacZ fusion to therpoS-dependent proP2 promoter (18Xu J. Johnson R.C. J. Bacteriol. 1995; 177: 5222-5231Google Scholar). Blue colonies in low salt media indicate ς38function, and white colonies are expected to contain nonfunctional ς38. These were subject to DNA sequence analysis.127 white colonies contained changes consisting solely of point substitutions (no frameshifts, insertions, deletions, or stop codons). Each mutant had from one to eight changes, with 21 colonies containing a single substitution and one colony yielding a plasmid with eight changes (Table I). Every position in the 50-amino acid stretch was changed at least once. The screen may have approached saturation as several of the mutants appeared more than once.Table IStatistical analysis of a nonfunctional ς38 (rpoS) libraryNo. of changes per colonyNo. of coloniesTotal No. of changes1212122652319574331325168061060717818Total number of colonies, 127; total number of changes, 417; number of amino acid positions, 50; expected number of changes/positions, 8.3 ± 4.2. Open table in a new tab Two approaches were taken to analyze the collection of nonfunctional mutants. In the first, a statistical analysis identified residues that were mutated most frequently in the library. This includes many plasmids that had lost ς38 function because of multiple mutations. Second, we simply identified single point substitutions that appeared in the library; only these were tested further using in vitro assays.Positions Commonly Mutated in Multiple Mutants in Region 2A statistical analysis of this non-functional library of substitutions was conducted (see “Experimental Procedures” and Table I) to identify amino acids that were mutated most frequently. Eight positions were found to be changed far more frequently than others (one standard deviation above the mean), suggesting they were selectively important for function (Fig. 1). We note that all of these eight positions have been shown to be important in ς70 function (Table II,top). For these positions a pair-wise analysis was conducted and showed that none were correlated strongly with mutations at other positions (data not shown). Accounting for codon usage (“hot spots”) by multivariate analysis did not alter the results significantly (data not shown).Table IIς38 positions non-randomly represented in the nonfunctional libraryOverrepresented positionsKnown propertiess38s70No. of changesPhe-140Tyr-42516Duplex binding in ς70 (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar)Trp-149Trp-43418Duplex binding in ς70(15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar)Arg-151Arg-43614Duplex binding in ς70 (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar)Gln-152Gln-43714Suppresses −12 mutation in ς70 (28Marr M.T. Roberts J.W. Science. 1997; 276: 1258-1260Google Scholar)Arg-156Arg-44113Duplex binding in ς70(15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar)Asn-160Asp-44514Binding and isomerization mutant in ς70 (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar)Ile-165Ile-45014Misfolded protein in ς70 (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar)Lys-173Glu-45818Supresses −14/−15 mutation in ς70 (30Barne K.A. Bown J.A. Busby S.J. Minchin S.D. EMBO J. 1997; 16: 4034-4040Google Scholar)Supresses −13 mutation in ς38 (5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar)Turnover element in ς38 (33Becker G. Klauck E. Hengge-Aronis R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6439-6444Google Scholar)Underrepresented positionsAla-130Ala-4154No informationTyr-145Tyr-4303Isomerization mutant in ς70 (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar, 16Tomsic M. Tsujikawa L. Panaghie G. Wang Y. Azok J. deHaseth P.L. J. Biol. Chem. 2001; 276: 31891-31896Google Scholar)Ile-158Ile-4434Partial core binding defect in ς70 (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar)Thr-164Thr-4492Minor duplex binding defect in ς70 (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar)Pro-168Pro-4531Partial core binding defect in ς70 (34Sharp M.M. Chan C.L. Lu C.Z. Marr M.T. Nechaev S. Merritt E.W. Severinov K. Roberts J.W. Gross C.A. Genes Dev. 1999; 13: 3015-3026Google Scholar)Glu-174Thr-4594No defect in ς38transcription (5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar)Auxillary role in ς38turnover (33Becker G. Klauck E. Hengge-Aronis R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6439-6444Google Scholar)Val-177Lys-4623No defect in ς38 transcription (5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar)Auxillary role in ς38 turnover (33Becker G. Klauck E. Hengge-Aronis R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6439-6444Google Scholar)Tyr-178Leu-4632No defect in ς38 transcription (5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar)The residue in ς38 (column one) and the analogous residue S.D. in ς70 (column two) are shown. The total number of times a particular residue was mutated in the library (column 3) and any previously identified phenotype associated with that residue is also shown (column 4). Overrepresented positions (top) are mutated more than one above the mean. Underrepresented positions (bottom) are mutated more than S.D. below the mean. Open table in a new tab Eight different positions were identified that appeared far less frequently than expected (one standard deviation below the mean; see Table II, bottom). Seven of these have been characterized previously with six having only minor to moderate defects, in either ς38 or ς70. This contrast supports the legitimacy of the screen and suggests that many of the same positions are important for both ςs.The most significant exception is Tyr-145 (Tyr-430 in ς70), which was identified previously as being critical for DNA melting and enzyme isomerization in ς70 (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar, 16Tomsic M. Tsujikawa L. Panaghie G. Wang Y. Azok J. deHaseth P.L. J. Biol. Chem. 2001; 276: 31891-31896Google Scholar). It is interesting to note that the Tyr-430 defects were identifiedin vitro and that mutations in this residue have not appeared in ς70 nonfunctional screens (23Waldburger C. Susskind M.M. J. Mol. Biol. 1994; 235: 1489-1500Google Scholar). Previous data also suggested that the defects were not as severe at high temperatures or on supercoiled DNA, both conditions present during the screen conducted here (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar, 24Juang Y.L. Helmann J.D. J. Mol. Biol. 1994; 235: 1470-1488Google Scholar).Single Point SubstitutionsThe importance of certain individual residues is derived more directly from white colonies containing plasmids with single substitutions. 18 such mutants (Fig. 1) were identified as non-functional (3 of the 21 colonies of this type contain redundant sequences). These mutations are spread fairly uniformly throughout the region. A minority of these correspond to positions of significant importance in ς70. The collection also includes three of the eight residues identified as overrepresented in the library (Phe-140, Trp-149, Gln-152). The lack of appearance of the other five residues could be because the screen did not reach saturation, or because they need to be coupled with other mutations to cause loss of function.These mutations depend on the loss of function of the proP2promoter. This promoter is activator-dependent and reaches its highest expression levels during the end of exponential phase (18Xu J. Johnson R.C. J. Bacteriol. 1995; 177: 5222-5231Google Scholar). The 18 point mutants were transferred to a differentrpoS − strain containing an aldBpromoter-lacZ fusion. This promoter does not require an activator for strong expression and is active during stationary phase (19Xu J. Johnson R.C. J. Bacteriol. 1995; 177: 3166-3175Google Scholar). 16 of the 18 mutants remained white on this screen indicating that these mutated residues were important for both promoters. The exceptions were R163H and R166H. We do not know the reason for the activity of these mutants at the aldB promoter. They also gave wild-type activity in subsequent primer extension and band shift experiments and are not discussed further. The remaining 16 point mutants identify amino acids important for ς38 function at both an activator-dependent and an activator-independent promoter.RNA LevelsThese mutants were analyzed further by isolating RNA from transformed cells and conducting primer extension using promoter-specific probes. None of the 16 point mutants gave a detectable signal for the aldB promoter (data not shown), consistent with the lack of function in the genetic screen. The same preparations were analyzed using a probe specific for dpsRNA. Upon entry into stationary phase, dps levels increase dramatically and are controlled at the level of transcription by ς38 (25Ali Azam T. Iwata A. Nishimura A. Ueda S. Ishihama A. J. Bacteriol. 1999; 181: 6361-6370Google Scholar, 26Lomovskaya O.L. Kidwell J.P. Matin A. J. Bacteriol. 1994; 176: 3928-3935Google Scholar). dps is a strong ς38 promoter, and mutated versions of ς38might be expected to show some function.Fig. 2 shows the results of comparingdps expression with that of the ς70 ampicillin RNA encoded by the plasmid. RNA levels were judged by comparing the strength of these dps andamp signals (Fig. 2 B). E132G, K133R, and M159T the only three mutants in which the dps signal was stronger than the amp signal (Fig. 2 A), had nearly wild-type activity. The defects associated with these mutant forms of ς38 can therefore be masked at a strong promoter.Figure 2A, primer extension of RNA from the ς38-dependent dps promoter. Vector (cells lacking ς38), wild-type ς38, and point mutants identified from the screen are labeled on thetop. AMP (arrow) represents the control signal from RNA expressed by the ampicillin gene.DPS (brackets) represents the signal from the ς38-dependent dps promoter. B, primer extension signals were quantified and standardized by taking the ratio of dps/ampicillin. Wild-type (W.t.) was set to the arbitrary value of 1.View Large Image Figure ViewerDownload (PPT)The remaining mutants showed a different pattern, and analysis indicated that their RNA levels were at least ∼4-fold down. The majority of the point mutants, therefore, remained significantly defective even at a strong promoter. This suggests that these 13 positions play a critical role in ς38-dependent transcription in vivo.Duplex DNA BindingDuplex DNA binding by these mutants was assessed using protein extracts and closed complex conditions. One complication is that ς38 holoenzyme binding to double strand DNA oligonucleotides in vitro is very weak (4Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Google Scholar). However, the presence of a −35 element can increase duplex binding significantly even though ς38 promoters do not typically contain −35 elements (6Gaal T. Ross W. Estrem S.T. Nguyen L.H. Burgess R.R. Gourse R.L. Mol. Microbiol. 2001; 42: 939-954Google Scholar). The addition of a −35 element to the synthetic fic con promoter strongly increased binding by ς38 holoenzyme in vitro (4Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Google Scholar). Using this template, crude protein extracts were used in EMSA experiments to test for duplex recognition (Fig. 3). The proteins were overexpressed, and levels were checked by SDS-PAGE (data not shown). All proteins migrated to the same position, and the amounts of protein were adjusted where necessary (see “Experimental Procedures”).Figure 3Closed complex binding to a duplex probe by holoenzymes containing mutant forms of ς38 as detected by EMSA. In typical experiments, 13% of the probe was bound by wild-type ς38; 18% binding by K133R; 4–11% by R129C, E132G, K133M, F140L, S143T, Q152R, and Y145A; 1–2% by W149R, M159T, I169T, and W148A; and undetectable levels by R141S, T153K, A157T, H170L, I171N, and L175P. The unbound probe is not shown.View Large Image Figure ViewerDownload (PPT)This assay showed high specificity for ς38 RNA polymerase (RNAP) binding as a strong band appeared using a vector overexpressing wt ς38 (top panel, lane 2) but did not appear from a vector without ς38(lane 1). This band runs at the same position as that seen by purified ς38 RNAP (data not shown). Binding by endogenous ςs, holoenzymes, or other DNA-binding proteins was not detected as no band appeared with the cells lacking ς38(lane 1). Occasionally, a band corresponding to nonspecific binding by core polymerase would appear (Fig. 3, left bottom panel, band below arrow), but this would be present in all lanes.The EMSA experiments were conducted at 4 °C to minimize the open conformation of the DNA and to limit nuclease and protease activity. None of the mutants showed an increase in nuclease or protease activity as compared with wild-type (data not shown). Thus the experiment primarily assays for closed complex formation. The use of crude cell extracts to test for DNA binding has also been used in the ς54 system (27Guo Y. Gralla J.D. J. Bacteriol. 1997; 179: 1239-1245Google Scholar).The 16 point mutants were overexpressed in E. coli, and crude cell extracts were prepared, mixed with purified core polymerase, and used in EMSA with a duplex fic con −35 probe (Fig. 3). Six mutants (R141S, T153K, A157T, H170L, I171N, L175P) did not give detectable levels of binding, likely accounting for their lack of function. Three mutants (W149R, M159T, I169T) were down ∼7-fold or more (Fig. 3), a fairly severe defect. The remaining seven mutants bound within 3-fold of wild-type. The only mutant with a wild-type level of binding was the conservative change K133R.We note that of the nine mutants that were down at least 7-fold, eight were in the 30-amino acid stretch C-terminal to Trp-149. By contrast, of the seven mutants with milder DNA binding defects, six were in the 20-amino acid stretch N-terminal to this residue. Thus it appears that the primary determinants for forming closed complexes with ς38 are in regions 2.4 and 2.5 with region 2.3 playing a different role.Properties of Two Site-directed MutantsTwo positions important for ς70 function, the conserved aromatics Y145A and W148A (Y430A and W433A in ς70) were not identified as non-functional in this screen. These play a major role in ς70 in vitro (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar, 16Tomsic M. Tsujikawa L. Panaghie G. Wang Y. Azok J. deHaseth P.L. J. Biol. Chem. 2001; 276: 31891-31896Google Scholar), although their rolein vivo is not known. Tyr-145 was also underrepresented in the library (Table II). Each of these was changed to alanine in ς38 (Fig. 1) and then characterized.Y145A and W148A were transferred to rpoS −strains carrying either a proP2 or aldBpromoter-lacZ fusion to test for ς38 functionin vivo. They were then screened using the blue/white colony test. Both site-directed mutants gave a light blue phenotype, indicating that they had partial function in vivo. Light blue colonies were not included in the screen of nonfunctional white colonies and thus Y145A and W148A would not have been identified. EMSA with duplex DNA (Fig. 3, right bottom panel), as described above, showed that both mutants led to a moderate defect in bindingin vitro, with Trp-148 showing the greater defect (4–8-fold).Core Polymerase BindingThe DNA binding experiments measure the interaction between holoenzyme and DNA. However, ς38requires core polymerase for binding, and so mutants that fail to bind core polymerase will also fail to bind DNA. We assayed the non-DNA binders for core binding using a ς competition protocol.In this assay radioactive DNA is mixed with wild-type ς38in the presence of a limiting amount of core polymerase. An excess of various mutant forms of ς38 are also present. If a mutant ς binds to core then it will titrate away the limiting core, leaving little of it associated with wild-type ς. Because neither mutant holoenzyme nor wild-type ς without core binds DNA (4Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Google Scholar), the radioactive DNA band should be diminished. To enhance sensitivity we used a very tight binding DNA fork junction probe; the weak binding mutants W149R, M159T, and I169T bind this probe, but the others do not. 2S. J. Lee and J. D. Gralla, unpublished data. The protein competition was applied to the other mutants, and some examples are shown in Fig. 4.Figure 4Competition binding experiments for various mutants on a single stranded fork probe that contains the start site as detected by EMSA. Lane 1 (W.t.) bound at 46% and lane 2 (4× W.t.) at 61%. Lane 3 andlane 5 bound at 20–32%; lanes 8, 10, and 12 bound at 48–59%; lanes 4 and6 bound at 5–8%; lane 7 bound at 38%;lanes 9 and 11 bound at 15–21%; and lane 13 bound at 64%.View Large Image Figure ViewerDownload (PPT)The four innermost pairs of lanes show that a 4-fold excess of different mutant proteins diminishes the signal, as expected from a protein that binds core but not DNA. An excess of two proteins that do bind DNA in holoenzyme form, wild-type and K133R, do not lead to a diminished signal (Fig. 4, outer pairs of lanes). Two of the proteins, T153K and L175P, gave signals that were less diminished, indicating that they bind polymerase but not as well as wild-type. We infer that all the non-DNA binders can bind core RNA polymerase, with two having a partial defect.Permanganate Assay for DNA OpeningA significant number of non-functional mutants showed relatively normal levels of binding DNA and core polymerase. These are expected be defective in steps subsequent to forming a closed promoter complex between holoenzyme and DNA. As open complex formation cannot be assayed with the short DNA probes used above, we turned to a plasmid-based system to explore potential defects in DNA melting by these mutants. Permanganate was used to assess whether the mutant holoenzymes were capable of forming open promoter complexes (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar, 20Sasse-Dwight S. Gralla J.D. J. Biol. Chem. 1989; 264: 8074-8081Google Scholar).Fig. 5 shows that all of these mutant holoenzymes exhibit defects in opening linearized plasmid promoter DNA. In all cases the melting signal is significantly less than that of wild-type, with most being only slightly higher than the background signal associated with DNA alone. These defects in opening appear to be significantly greater than the minor decreases seen in duplex binding by these same mutants (Fig. 3). We infer that the residues within this region play a significant role in melting the DNA after polymerase forms a closed complex.Figure 5KMnO4 probing of Escherichia colimutant ς38 holoenzymes on the fic conpromoter template strand. Controls with core alone and wild-type ς38 holoenzyme are on the far left side. Reactions were performed at 30 °C on linearized plasmid DNA.View Large Image Figure ViewerDownload (PPT)DISCUSSIONVery little is known about how ς38 accomplishes promoter recognition. A prior study implicated region 2.5 of ς38 as being important for recognition of sequences upstream from the −7 to −12 DNA sequence element (5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar). Other inferences about recognition use analogies with ς70, which is 60% identical to ς38 in its potential DNA recognition regions 2.3, 2.4, and 2.5 (see Fig. 2). The role of the conserved and non-conserved amino acids is unknown as mutations in regions 2.3 and 2.4 of ς38 have not been identified. In this work we identify 22 positions (20 screened and 2 site-directed mutants) in region 2 that affect ς38 function. Below we use these data to locate the important regional determinants of ς38 function and use the large base of available structural and mutational data on ς70 to draw additional conclusions about the two proteins.Binding of ς38 to Duplex DNAImportant positions were identified as sites of single substitutions or from a statistical analysis of clones containing multiple mutations. The single substitutions were spread fairly evenly throughout most of regions 2.3, 2.4, and 2.5.The single substitutions were assayed for DNA binding using duplex DNA under closed complex conditions. When the results were interpreted in terms of location an interesting pattern emerged. Nearly all (eight of nine) of the most defective mutants were C-terminal to position Trp-149. Those with milder defects (six of seven) were N-terminal to Trp-149. From this distribution we infer that the determinants of closed complex formation are primarily in regions 2.4 and 2.5. Region 2.3 is also clearly important, as judged from the mutational analysis, but its main function does not appear to be recognition of duplex DNA to form closed complexes. Some of the positions in these and other regions may not be involved directly in ς38 function but could instead alter the local structure of the protein. Gross misfolding is unlikely as all mutants appear to bind core to form holoenzyme.Comparison with ς70 and ImplicationsOrganization and Residuesς38 and ς70 recognize very similar but not identical DNA sequences near the downstream −10 promoter element (4Lee S.J. Gralla J.D. J. Biol. Chem. 2001; 276: 30064-30071Google Scholar, 5Becker G. Hengge-Aronis R. Mol. Microbiol. 2001; 39: 1153-1165Google Scholar, 6Gaal T. Ross W. Estrem S.T. Nguyen L.H. Burgess R.R. Gourse R.L. Mol. Microbiol. 2001; 42: 939-954Google Scholar). The proteins are highly homologous in the regions just discussed. Therefore comparison of what is known about the two ςs should reveal new information about both.The overall functional organization appears to be similar in the two ςs. Several studies of ς70 place Trp-148, Trp-149 and Gln-152 at or near the −12/−11 fork junction boundary between single and double strand DNA (15Fenton M.S. Lee S.J. Gralla J.D. EMBO J. 2000; 19: 1130-1137Google Scholar, 28Marr M.T. Roberts J.W. Science. 1997; 276: 1258-1260Google Scholar). The C-terminal segment is thought to interact with double strand DNA, and the N-terminal segment is thought to interact largely with non-duplex DNA. This is essentially the same arrangement inferred above for ς38.Despite this similarity of arrangement, the residu" @default.
• W2061430251 created "2016-06-24" @default.
• W2061430251 creator A5043656920 @default.
• W2061430251 creator A5059596664 @default.
• W2061430251 date "2002-12-01" @default.
• W2061430251 modified "2023-09-30" @default.
• W2061430251 title "Promoter Use by ς38 (rpoS) RNA Polymerase" @default.
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