Journal of Bacteriology, December 2000, p. 6774-6782, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045
Received 21 August 2000/Accepted 18 September 2000
The Escherichia coli rhaSR operon encodes two AraC
family transcription activators, RhaS and RhaR, and is activated by
RhaR in the presence of L-rhamnose.
-Galactosidase assays of various rhaS-lacZ promoter
fusions combined with mobility shift assays indicated that a cyclic AMP
receptor protein (CRP) site located at
111.5 is also required for
full activation of rhaSR expression. To address the
mechanisms of activation by CRP and the RNA polymerase
-subunit
C-terminal domain (
-CTD) at rhaSR, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of
(
-
235), and alanine
substitutions throughout
-CTD. We found that DNA-contacting residues
in
-CTD are required for full activation, and for simplicity, we
discuss
-CTD as a third activator of rhaSR. CRP and RhaR
could each partially activate transcription in the absence of the other
two activators, and
-CTD was not capable of activation alone. In the
case of CRP, this suggests that this activation involves neither an
-CTD interaction nor cooperative binding with RhaR, while in the
case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and
-CTD each have synergistic effects on activation by the others, suggesting direct
or indirect interactions among all three. We have some evidence that
the
-CTD-CRP and
-CTD-RhaR interactions might be direct. The
magnitude of the synergistic effects was usually greater with just two
activators than with all three, suggesting possible redundancies in the
mechanisms of activation by CRP,
-CTD, and RhaR.
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