The importance of a proper helical structure in the promoter-10 binding region to Bacillus subtilis sigma A structure and function.

Abstract

Two Bacillus subtilis sigA mutants with amino acid substitutions tending to disrupt the structure of the promoter -10 binding helix of B. subtilis sigma A factor were constructed. B. subtilis DB1001 which contained an A197P substitution was very sensitive to temperature elevation. B. subtilis DB1002 had a T199G substitution and was low in growth potential at the elevated temperature. Degradation of sigma A in B. subtilis DB1001 (t(1/2)=63.2 min) and DB1002 (t(1/2)=186.0 min) occurred readily even at 37 degrees C; however, sigma A in B. subtilis DB2 (wild-type) was fairly stable at the same temperature. The activities of both DB1001 and DB1002 sigma A factors on groE promoter (sigma A-type) were lower than those of the wild-type counterpart at both permissive and restrictive temperatures. The failure of a higher sigma A concentration to suppress the Ts phenotype of DB1001 indicated that the temperature sensitivity of B. subtilis DB1001 was due to altered function, rather than insufficient concentration, of sigma A in the cells. Taken together, our results suggest that the helicity of the promoter -10 binding helix is essential to the packing interaction in the hydrophobic core region of sigma A, which helps to maintain the stable and functional sigma A structure.