SEQUENCE-SPECIFIC INTERACTIONS BETWEEN PROMOTER DNA AND THE RNA-POLYMERASE SIGMA-FACTOR-E

In order to determine which amino acyl residues in a secondary sigma factor govern its specificity of recognition at the -35 region of promoters, we examined the effects of amino acid substitutions in sigma(E) in Bacillus subtilis that made the sequence of its putative -35 recognition region more similar to another sigma factor in B. subtilis, sigma(K). We found that a single amino acid substitution at position 217 of sigma(E) resulted in a sigma factor that could direct transcription from sigma(K)-dependent promoters. Furthermore, we tested whether this amino acid substitution in sigma(E) had changed the specificity of interactions of the sigma with -35 region sequences by examining the activity of the mutant sigma(E) on derivatives of sigma(E)-dependent promoters that contained single base-pair substitutions. We found that this substitution in sigma(E) specifically suppressed the effect of a single base-pair substitution at position -31 in a sigma(E)-dependent promoter spoIIID. The amino acyl residue at another position (219) in sigma(E) affected the specificity of interaction with position -33 in spoIIID promoter. The amino acyl residues at the two positions in sigma(E), 217 and 219, that determine the specificity of interactions between the sigma and base-pairs in the -35 region of its cognate promoters (positions -33 and -31, respectively, in the spoIIID promoter) probably closely contact these base-pairs. (C) 1995 Academic Press Limited