title: Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C.
abstract: 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.
authors.affiliation: Department of Biology, University of Rochester, NY 14627, USA.
authors.forename: W
authors.initials: W
authors.lastname: Sun
authors.forename: S
authors.initials: S
authors.lastname: Hattman
mesh_terms: D001426:Bacterial Proteins / Q000494:pharmacology; D010583:Bacteriophage mu / Q000187:drug effects; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology; D029968:Escherichia coli Proteins; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics / Q000494:pharmacology*; D015533:Transcriptional Activation; D001616:beta-Galactosidase
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C499299:oxyR protein, E coli; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase