Nucleic Acids Research, Vol 27, Issue 23 4541-4546, Copyright © 1999 by Oxford University Press
J Qiu and JD Helmann
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.
ARTICLES
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
Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
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