Effect of mutations in the "extended -10" motif of three Bacillus subtilis σA-RNA polymerase-dependent promoters

The "extended -10" motif described originally in Escherichia coli promoters occurs frequently in other bacterial promoters. Most Bacillus subtilis bacteriophage empty set 29 promoters contain this motif. To analyse the influence of the motif on sigma(A)-RNA polymerase transcription, the 5'-TG-3' dinucleotide was changed to 5'-AC-3' in three empty set 29 promoters. This change impaired RNA polymerase binding to the promoters; the yields of closed and open complexes were reduced independently of other differences inherent to each promoter. The mutation abolished transcription in vitro from a promoter lacking the consensus sequence at the -35 hexamer. In contrast, at other promoters with a -35 consensus sequence, the yield of run off transcription was not reduced by the mutation. Indeed an apparent interference phenomenon at high polymerase/DNA ratios was relieved. These results indicate that the extended -10 motif provides contact points for sigma(A)-RNA polymerase with a role restricted to the first steps of transcription. (C) 1999 Academic Press.