Nucleic Acids Research, Vol 24, Issue 12 2242-2251, Copyright © 1996 by Oxford University Press
TA Belyaeva, JA Bown, N Fujita, A Ishihama and SJ Busby
Hydroxyl radical footprinting has been used to study different open
complexes between Escherichia coli RNA polymerase and the galactose operon
regulatory region, which contains two overlapping promoters, P1 and P2.
Complexes at P1 were studied by exploiting a P2- mutant and complexes at P2
were studied with a P1-mutant. We have identified the precise location of
alpha binding in both binary RNA polymerase-galP1 and RNA polymerase-P2
complexes from the effects of deletion of the C- terminal domain of the RNA
polymerase alpha subunit: alpha binds to different sites at the upstream
end of each complex. Transcription initiation at galP1 can be activated by
the cyclic AMP receptor protein (CRP). Addition of CRP to the RNA
polymerase-galP1 complex displaces the C-terminal domain of alpha, which
then binds to a different site upstream of CRP in the ternary CRP-RNA
polymerase-galP1 complex. Thus, the C-terminal domain of alpha can occupy
three different sites at the gal operon regulatory region. We have also
examined the effect of disrupting the Activating Region of CRP on
interactions between CRP and the C-terminal domain of alpha in ternary
CRP-RNA polymerase-galP1 complexes. Footprinting experiments show that
these substitutions interfere with the contact between CRP and alpha but do
not affect the position of alpha binding to its site upstream of bound CRP.
ARTICLES
Location of the C-terminal domain of the RNA polymerase alpha subunit in different open complexes at the Escherichia coli galactose operon regulatory region
School of Biochemistry, University of Birmingham, UK.
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