Nucleic Acids Research, Vol 24, Issue 20 4042-4049, Copyright © 1996 by Oxford University Press
W Sun and S Hattman
Transcription of the bacteriophage Mu mom operon requires transactivation
by the phage-encoded C protein. DNase I footprinting showed that in the
absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to
the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect
to the transcription start site), on the top (non-transcribed) strand. This
is slightly upstream from, but overlapping P1 (-49 to +16), the functional
binding site for rightward transcription. Host DNA-[N6-adenine]
methyltransferase (Dam) methylation of three GATCs immediately upstream of
the C binding site is required to prevent binding of the E.coli OxyR
protein, which represses mom transcription in dam- strains. OxyR, known to
induce DNA bending, is normally in a reduced conformation in vivo, but is
converted to an oxidized state under standard in vitro conditions. Using
DNase I footprinting, we provide evidence supporting the proposal that the
oxidized and reduced forms of OxyR interact differently with their target
DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to
repress mom expression in vivo in a dam- host. In vitro DNase I
footprinting showed that OxyR-C199S protected Pmom from - 104 to -46 on the
top strand and produced a protection pattern characteristic of reduced
wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to
P2 (in the absence of C), whereas it only slightly decreased binding of C
to its target site (-55 to -28, as defined by DNase I footprinting). In
contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to
P1. These results indicate that OxyR repression is mediated subsequent to
binding by C. Mutations have been isolated that relieve the dependence on C
activation and have the same transcription start site as the C-activated
wild-type promoter. One such mutant, tin7, has a single base change at -14,
which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP
binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1
binding sites probably do not overlap, and in vivo expression of tin7 was
reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can
repress tin7.
ARTICLES
Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C
Department of Biology, University of Rochester, NY 14627, USA.
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