Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase.
Coliphage N4 virion RNA polymerase (vRNAP), the most distantly related member of the T7-like family of RNA polymerases, is responsible for transcription of the early genes of the linear double-stranded DNA phage genome. Escherichia coli single-stranded DNA-binding protein (EcoSSB) is required for N4 early transcription in vivo, as well as for in vitro transcription on super-coiled DNA templates containing vRNAP promoters. In contrast to other DNA-dependent RNA polymerases, vRNAP initiates transcription on single-stranded, promoter-containing templates with in vivo specificity; however, the RNA product is not displaced, thus limiting template usage to one round. We show that EcoSSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling. EcoSSB binds to the template and to the nascent transcript and prevents the formation of a transcriptionally inert RNA:DNA hybrid. Using C-terminally truncated EcoSSB mutant proteins, human mitochondrial SSB (Hsmt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hsmt-EcoSSB chimera, we have mapped a determinant of template recycling to the C-terminal amino acids of EcoSSB. T7 RNAP contains an amino-terminal domain responsible for binding the RNA product as it exits from the enzyme. No sequence similarity to this domain exists in vRNAP. Hereby, we propose a unique role for EcoSSB: It functionally substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding.
Davydova
Elena K
EK
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
Rothman-Denes
Lucia B
LB
D000595:Amino Acid Sequence; D025522:Bacteriophage N4 / Q000201:enzymology*; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases