Orientation of OmpR monomers within an OmpR:DNA complex determined by DNA affinity cleaving.
Escherichia coli OmpR is a transcription factor that regulates the differential expression of the porin genes ompF and ompC. Phosphorylated OmpR binds as a dimer to a 20-bp region of DNA consisting of two tandemly arranged 10-bp half-sites. Expression of the ompF gene is achieved by the hierarchical occupation of three adjacent 20-bp binding sites, designated F1, F2, and F3 and a distally located site, F4. Despite genetic, biochemical, and structural studies, specific details of the interaction between phosphorylated OmpR and the DNA remain unknown. We have linked the DNA cleaving moiety o-phenanthroline-copper to eight different sites within the DNA binding domain of OmpR in order to determine the orientation of the two OmpR monomers in the OmpR:F1 complex. Five of the resulting conjugates exhibited DNA cleaving activity, and four of these yielded patterns that could be used to construct a model of the OmpR:F1 complex. We propose that OmpR binds asymmetrically to the F1 site as a tandemly arranged dimer with each monomer having its recognition helix in the major groove. The N-terminal end of the recognition helix is promoter-proximal and flanked by "wings" W1 and W2 positioned proximally and distally, respectively, to the transcription start site of ompF. We further propose that the C-terminal end of the recognition helix makes the most extensive contacts with DNA and predict bases within the F1 site that are sufficiently close to be contacted by the recognition helix.
Harrison-McMonagle
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P
Center for Advanced Biotechnology and Medicine, 679 Hoes Ln, Piscataway, NJ, 08854, USA.
Denissova
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N
Martínez-Hackert
E
E
Ebright
R H
RH
Stock
A M
AM
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018272:Porins / Q000235:genetics; D011487:Protein Conformation; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C109237:OmpF protein; D018272:Porins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D003850:Deoxyribonuclease I