Adenines at-11,-9 and-8 play a key role in the binding of Bacillus subtilis EσA RNA polymerase to-10 region single-stranded DNA

The sigma subunit of RNA polymerase interacts with the promoter DNA in at least two regions: the -35 and the -10 consensus elements. The latter contacts are involved both in recognition and in melting of the promoter DNA to form the transcriptionally-competent open complex. RNA polymerase holoenzyme, but neither core nor sigma alone, binds with high selectivity to single-stranded DNA (ssDNA) containing the nontemplate -10 consensus sequence. We have used equilibrium competition to assess the specificity of holoenzyme binding to a 19 base oligonucleotide containing a -10 consensus element, TATAAT, Analysis of all 18 possible single point mutations in the -10 consensus sequence reveals that binding by Bacillus subtilis E sigma(A) holoenzyme depends critically upon adenine at position -11 and, unexpectedly, is strongly affected by substitutions of the poorly conserved adenines at -9 and -8. Similarly, ssDNA binding by Escherichia coli E sigma(70) holoenzyme is most strongly affected by substitutions of adenines within the -10 region consensus. The critical role of -11A in binding ssDNA supports a key role for this base in the nucleation of DNA melting. A novel role for -9A and -8A is proposed in the context of recent models of promoter melting.