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 f actor govern its specificity of recognition at the -35 region of promo ters, we examined the effects of amino acid substitutions in sigma(E) in Bacillus subtilis that made the sequence of its putative -35 recogn ition region more similar to another sigma factor in B. subtilis, sigm a(K). We found that a single amino acid substitution at position 217 o f 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 in teractions of the sigma with -35 region sequences by examining the act ivity of the mutant sigma(E) on derivatives of sigma(E)-dependent prom oters that contained single base-pair substitutions. We found that thi s substitution in sigma(E) specifically suppressed the effect of a sin gle base-pair substitution at position -31 in a sigma(E)-dependent pro moter spoIIID. The amino acyl residue at another position (219) in sig ma(E) affected the specificity of interaction with position -33 in spo IIID 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