Interactions of Bacillus subtilis RNA polymerase with subunits determining the specificity of initiation. Sigma and delta peptides can bind simultaneously to core

The Bacillus subtilis RNA polymerase sigma 43 subunit and the phage SP82 encoded 28-kDa peptide are responsible for the binding of RNA polymerase to early and middle SP82 promoters, respectively. The delta peptide enhances the specificity of the interaction of B. subtilis RNA polymerase with these promoters. We have used sedimentation experiments to determine the effect of each of the three specificity factors, delta, sigma, and the 28-kDa peptide, on the binding of the other two factors to RNA polymerase core and the effect of NaCl on these binding equilibria. We show that sigma 43 and the 28-kDa peptide can each bind to RNA polymerase core at the same time as delta. Sigma 43 and the 28-kDa peptide have similar affinities to core at 0.1 M NaCl, but the 28-kDa peptide binds to core-delta more strongly than sigma 43. The implications of these findings with respect to the replacement of sigma 43 by the 28-kDa peptide and the mechanism of promoter search by B. subtilis RNA polymerase are discussed.     

Peptide mapping of Bacillus subtilis RNA polymerase alpha factors and core-associated polypeptides

An analysis of the peptide maps of the sigma factors and core-associated subunits of Bacillus subtilis RNA polymerase has revealed that all the sigma factors ad core-associated polypeptides are derived from separate genes and are not proteolytically modified products of the major 55,000-dalton sigma factor. A comparison of the peptide pattern of the major B. subtilis and Escherichia coli sigma factors revealed limited homology between them. Furthermore, antibody prepared against the 55,000-dalton B. subtilis sigma factor cross-reacted against the E. coli sigma factor, but not against any of the other B. subtilis sigma factors and core-associated polypeptides. These results unambiguously demonstrate the independently derived nature of the B. subtilis RNA polymerase core-associated subunits and the partial relationship between the major sigma factors of B. subtilis and E. coli.     

Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters.

The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage lambda promoters lambda pR and lambda pL. The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat. In vitro, the relative affinities of purified sigma 70-RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from lambda pR that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original lambda pR. Binding of E. coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis. Both the supercoiled and relaxed forms of the hybrid lambda pL series were better competitors for E. coli RNA polymerase binding than was the original lambda pL. The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid lambda pR promoters around the E. coli RNA polymerase in a tight, nucleosomal-like fashion. The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.     

Changes in the association between Bacillus subtilis RNA polymerase core and two specificity-determining subunits during transcription

The Bacillus subtilis RNA polymerase sigma subunit and the phage SPO1-coded gene 28 protein are responsible for selective binding of RNA polymerase to early and middle SPO1 promoters, respectively. The association of the RNA polymerase core with each of these subunits weakens during the elongation of RNA chains. Similar changes are known to be an essential part of the Escherichia coli RNA polymerase sigma cycle.     

A single amino acid substitution in sigma E affects its ability to bind core RNA polymerase.

We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis. sigma E is required for the production of endospores in B. subtilis but not for vegetative growth. Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores. Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase. The substitution at position 68 did not affect the stability of the protein in B. subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor. The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.     

Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins.

We show, using dot matrix comparisons and statistical analysis of sequence alignments, that seven sequenced sigma factors, E. coli sigma-70 and sigma-32, B. subtilis sigma-43 and sigma-29, phage SP01 gene products 28 and 34, and phage T4 gene product 55, comprise a homologous family of proteins. Sigma-70, sigma-32, and sigma-43 each have two copies of a sequence similar to the helix-turn-helix DNA binding motif seen in CRP, and lambda repressor and cro proteins. B. subtilis sigma-29, SP01 gp28, and SP01 gp34 have at least one copy similar to this sequence. We propose that a second sequence, conserved in all seven proteins is the core RNA polymerase binding site. A third region, present only in sigma-70 and sigma-43, may also be involved in interaction with core. Available mutational evidence supports our model for sigma factor structure.