PMID:12876194
| Citation |
Davydova, EK and Rothman-Denes, LB (2003) Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 100:9250-5 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC170904 Online version:10.1073/pnas.1133325100 |
| Keywords |
Amino Acid Sequence; Bacteriophage N4/enzymology; Base Sequence; Cross-Linking Reagents/pharmacology; DNA/metabolism; DNA-Binding Proteins/chemistry; DNA-Directed RNA Polymerases/chemistry; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli/metabolism; Humans; Mitochondria/metabolism; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; RNA/metabolism; Recombinant Fusion Proteins/metabolism; Sequence Homology, Amino Acid; Time Factors; Transcription, Genetic |
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