Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C

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 (P-mom) 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 Pt (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N-6-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 farms 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 P-mom 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 T-6 run to T(3)GT(2). 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.