GENETIC AND PHYSIOLOGICAL-STUDIES OF BACILLUS-SUBTILIS SIGMA(A) MUTANTS DEFECTIVE IN PROMOTER MELTING

The Bacillus subtilis sigA gene encodes the primary sigma factor of RN A polymerase and is essential for cell growth. We have mutated conserv ed region 2.3 of the sigma(A) protein to substitute each of seven arom atic amino acids with alanine. Several of these aromatic amino acids a re proposed to form a melting motif which facilitates the strand separ ation step of initiation. Holoenzymes containing mutant sigma factors recognize promoters, but some are defective for DNA melting in vitro. We have studied the ability of each mutant sigma factor to support cel l growth by gene replacement and complementation. The two region 2.3 m utants least impaired in promoter melting in vitro (Y180A and Y184A) s upport cell growth in single copy, although the Y184A allele imparts a slow-growth phenotype at low temperatures. A strain expressing only t he Y189A variant of the sigma(A) protein, known to be defective in DNA melting in vitro, grows very slowly and is altered in its pattern of protein synthesis. Only the wild-type and Y180A sigma(A) proteins effi ciently complement a temperature-sensitive allele of sigA. Overexpress ion of three of the sigma(A) proteins defective for promoter melting i n vitro (Y189A, W192A, and W193A) leads to a decrease in RNA synthesis and cell death. These results indicate that mutations which specifica lly impair DNA melting in vitro also impair sigma function in vivo and therefore support the hypothesis that sigma plays an essential role i n both DNA melting and promoter recognition.