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

Nucleic Acids Res. 1999 Dec 1;27(23):4541-6. doi: 10.1093/nar/27.23.4541.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine / metabolism*
  • Bacillus subtilis / enzymology*
  • Base Sequence
  • DNA, Single-Stranded / metabolism*
  • DNA-Directed RNA Polymerases / chemistry
  • DNA-Directed RNA Polymerases / metabolism*
  • Protein Binding
  • Sigma Factor / chemistry
  • Sigma Factor / metabolism*

Substances

  • DNA, Single-Stranded
  • Sigma Factor
  • RNA polymerase sigma A
  • DNA-Directed RNA Polymerases
  • Adenine