Nucleic Acids Research, Vol 25, Issue 13 2603-2609, Copyright © 1997 by Oxford University Press
X Huang, FJ Lopez de Saro and JD Helmann
Thesigmasubunit of RNA polymerase determines promoter recognition and
catalyzes DNA strand separation. The -35 promoter region is recognized by a
helix-turn-helix motif in region 4, while the -10 region is specified, at
least in part, by an amphipathic helix in region 2. We have proposed that
conserved aromatic residues insigmaregion 2.3 interact with the
non-template strand of the -10 element to drive open complex formation. We
now report that Bacillus subtilis sigmaA holoenzyme, but neither core nor
sigmaA alone, binds with high selectivity to single-stranded (ss) DNA
containing the non-template -10 consensus sequence. UV irradiation of
holoenzyme-ssDNA complexes efficiently crosslinks sigmaA to DNA and
protease mapping supports a primary contact site in or near region 2.
Several mutations in sigmaA region 2.3, shown previously to impair promoter
melting, affect ssDNA binding: Y184A decreases binding selectivity, while
Y189A and W193A decrease the efficiency of photocrosslinking. These results
support a model in which these aromatic amino acids are juxtaposed to
ssDNA, consistent with their demonstrated role in stabilizing the open
complex.
ARTICLES
sigma factor mutations affecting the sequence-selective interaction of RNA polymerase with -10 region single-stranded DNA
Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
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