Sequence and DNA structural determinants of N4 virion RNA polymerase-promoter recognition.
Coliphage N4-coded, virion-encapsidated RNA polymerase (vRNAP) is able to bind to and transcribe promoter-containing double-stranded DNAs when the template is supercoiled and Escherichia coli single-stranded DNA-binding protein (Eco SSB) is present. We report that vRNAP-promoter recognition and activity on these templates require specific sequences and a hairpin structure on the template strand. Hairpin extrusion, induced by Mg(II) and physiological superhelical density, is essential to provide the correct DNA structure for polymerase recognition, as mutant promoters that do not form hairpins show reduced in vitro activity. Therefore, a supercoil-induced DNA structural transition regulates N4 vRNAP transcription. Eco SSB activates transcription at physiological superhelical densities by stabilizing the template-strand hairpin. Specific sequences at the promoters are conserved to provide proper contacts for vRNAP, to support hairpin extrusion, or both. We propose a model for in vivo utilization of the vRNAP promoters, and discuss the roles of DNA supercoiling and Eco SSB in promoter activation.
Dai
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Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Rothman-Denes
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D001483:Base Sequence; D001665:Binding Sites; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017902:Podoviridae / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004278:DNA, Superhelical; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases