THE BACILLUS-SUBTILIS FLAGELLAR REGULATORY PROTEIN SIGMA(D) - OVERPRODUCTION, DOMAIN ANALYSIS AND DNA-BINDING PROPERTIES

Flagellar biosynthesis requires an alternative sigma (sigma) subunit o f RNA polymerase to allow recognition of the promoters for flagellin a nd other late genes of the flagellar regulon. We have now overproduced and characterized Bacillus subtilis sigma(D): the prototype of the si gma(28) family of flagellar a factors. Limited protease digestion stud ies indicate that sigma(D) contains an amino-terminal domain, comprisi ng conserved regions 1.2 and 2, and a carboxyl-terminal domain contain ing conserved regions 3.2 and 4. The protease-sensitive region between these two domains correlates with a region of very low sequence conse rvation among bacterial sigma factors. Unlike the primary sigma factor , sigma(D) binds to DNA. In non-denaturing polyacrylamide gel electrop horesis the sigma(D)-DNA complex has an apparent equilibrium dissociat ion constant of 1 mu M. Binding of sigma(D) to the promoter for flagel lin, P-D-6, appears to lead to an altered DNA structure near the -35 a nd -10 recognition elements as detected by DNase I footprinting and by the enhanced reactivity of several bases to dimethylsulfate.