Interchangeability of delta subunits of RNA polymerase from different species of the genus Bacillus.

RNA polymerase was purified from five species of Bacillus, including Bacillus subtilis. Each polymerase had a subunit composition analogous to that reported for B. subtilis, i.e., beta beta '2 alpha sigma delta omega 1 omega 2. The delta subunits from the B. subtilis and Bacillus thuringiensis polymerases were interchangeable, as judged from their effects on promoter selection in the polymerase binding assay.     

Purification and properties of RNA polymerases from mother cells and forespores of sporulating cells of Bacillus subtilis

Bacillus subtilis sporulating cells at stage III were fractionated into mother cell and forespore fractions by means of a lysozyme-detergent method. Three forms of DNA-dependent RNA polymerase enzymes, termed M sigma, F sigma, and F delta, in addition to core enzyme (alpha 2, beta', and beta) have been purified from the cell fractions. Enzymes M sigma and F sigma are present in the mother cell and forespore, respectively, and contain sigma factor of 55,000 daltons in addition to the core subunits. On the other hand, enzyme F delta is present specifically in the forespore and contains delta 1 factor of 28,000 daltons instead of the sigma factor. The amount of RNA polymerase in the forespore is about twice that in the mother cell. The enzymes M sigma and F sigma also differed in their elution profiled from DEAE-cellulose columns and in their heat stabilities indicating that the two sigma-containing holoenzyme forms may be different in their structural properties. The enzyme F delta transcribed B. subtilis DNA about 1.6 times more actively than enzyme F sigma, and the enzymes M sigma and F sigma transcribed the DNA about 2.2 times more actively than did core enzyme.     

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.     

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.