Adenines at -11, -9 and -8 play a key role in the binding of Bacillus subtilis Esigma(A) 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 non-template -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 Esigma(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 Esigma(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.
Qiu
J
J
Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
Helmann
J D
JD
D000225:Adenine / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*
D004277:DNA, Single-Stranded; D012808:Sigma Factor; C063505:RNA polymerase sigma A; D012321:DNA-Directed RNA Polymerases; D000225:Adenine