Open complex formation in vitro by sigma38 (rpoS) RNA polymerase: roles for region 2 amino acids

Non-functional mutants of sigma(38)(sigma(S)) were studied in vitro to identify the nature of their defects. Mutations in four amino acids led to severe defects in DNA binding and enzyme isomerization with promoter fork junction probes containing single-stranded non-template DNA. The same properties were previously seen with DNA mutations at the fork junction, implying that sigma:DNA interactions at the fork junction are used both for DNA binding and enzyme isomerization. An overlapping set of four mutants had defects that appear to be associated with DNA melting to create the fork junction. When mapped onto the sigma(70) structure, these groups of mutants suggest motifs used by sigma factors to melt DNA and isomerize RNA polymerase to form functional open promoter complexes.     

Different roles for basic and aromatic amino acids in conserved region 2 of Escherichia coli sigma(70) in the nucleation and maintenance of the single-stranded DNA bubble in open RNA polymerase-promoter complexes.

Amino acid residues in region 2 of final sigma(70) have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with single-stranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the -10 region. We concluded that two basic amino acids (Lys(414) and Lys(418)) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr(430) and Trp(433)). It is likely that these four amino acids, which are close to each other in the structure of final sigma(70), together are involved in the nucleation of the strand separation process.     

Sigma38 (rpoS) RNA polymerase promoter engagement via -10 region nucleotides

Band shift assays using DNA probes that mimic closed and open complexes were used to explore the determinants of promoter recognition by sigma38 (rpoS) RNA polymerase. Duplex recognition was found to be much weaker than that observed in sigma70 promoter usage. However, binding to fork junction probes, which attempt to mimic melted DNA, was very strong. This binding occurs via the non-template strand with the identity of the two conserved junction nucleotides (-12T and -11A) being of paramount importance. A modified promoter consensus sequence identified these two nucleotides as among only four (underlined) that are highly conserved, and all four were in the -10 region (CTAcacT from -13 to -7). The remaining two nucleotides were shown to have different roles, -13C in preventing recognition by the heterologous sigma70 polymerase and -7T in directing enzyme isomerization. These -10 region nucleotides appear to have their primary function prior to full melting because probes that had a melted start site were relatively insensitive to substitution at these positions. These results suggest the sigma38 mechanism differs from the sigma70 mechanism, and this difference likely contributes to selective use of sigma38 under conditions that exist during stationery phase.     

DNA-binding determinants of sigma 54 as deduced from libraries of mutations.

PCR mutagenesis was used to obtain libraries of mutations in the region between amino acids 300 and 400 in the DNA-binding domain of Escherichia coli sigma 54. Two hundred changes that did not alter function were identified. These were compared with a somewhat smaller number of changes that did alter function. Several important regions were identified. Single point mutations in two of these, near amino acids 363 and 383, destroyed the ability of sigma to bind DNA, as assayed by band shift analysis. A third segment from amino acids 327 to 347 is also a candidate for contributing to DNA binding. Comparison with data in the literature leads to testable proposals for the complex mode of DNA binding that is associated with sigma 54.     

PCR mutagenesis identifies a polymerase-binding sequence of sigma 54 that includes a sigma 70 homology region.

Sigma 54 is a minor bacterial sigma factor that is not a member of the sigma 70 family of proteins but binds the same core RNA polymerase. Previously, we identified a region of sigma 54 that is important for binding core polymerase. In this work, PCR mutagenesis was used to identify specific amino acids important for this binding. The results show that important residues are clustered most closely in a short sequence that was previously speculated to be potentially homologous to a sequence in sigma 70. The mutagenesis also identifies important residues in the flanking hydrophobic-acidic region of sigma 54, which is absent in sigma 70. Overall, the data indicate that sigma 54 binds core polymerase through a sequence homologous to that of sigma 70 but in addition uses unique motifs to modify this interaction.     

Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions.

By a quantitative Western immunoblot analysis, the intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma S, the rpoS gene product), and of two minor sigma subunits, sigma 54 (sigma N, the rpoN gene product) and sigma 28 (sigma F, the rpoF gene product), were determined in two Escherichia coli strains, W3110 and MC4100. The results indicated that the levels of sigma 54 and sigma 28 are maintained at 10 and 50%, respectively, of the level of sigma 70 in both strains growing at both exponential and stationary phases, but in agreement with the previous measurement for strain MC4100 (M. Jishage and A. Ishihama, J. Bacteriol. 177:6832-6835, 1995), the level of sigma 38 was undetectable at the exponential growth phase but increased at 30% of the level of sigma 70 at the stationary phase. Stress-coupled change in the intracellular level was observed for two sigma subunits: (i) the increase in sigma 38 level and the decrease in sigma 28 level upon exposure to heat shock at the exponential phase and (ii) the increase in sigma 38 level under high-osmolality conditions at both the exponential and stationary phases.