Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
| Autor: | Elena K. Davydova, Lucia B. Rothman-Denes |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Time Factors
Transcription Genetic Recombinant Fusion Proteins Molecular Sequence Data RNA-dependent RNA polymerase chemistry.chemical_compound Transcription (biology) RNA polymerase Bacteriophage N4 Escherichia coli Humans Amino Acid Sequence Promoter Regions Genetic Gene Polymerase Multidisciplinary Base Sequence Dose-Response Relationship Drug Sequence Homology Amino Acid biology RNA Promoter DNA DNA-Directed RNA Polymerases Biological Sciences Molecular biology Mitochondria Protein Structure Tertiary Cell biology DNA-Binding Proteins stomatognathic diseases Cross-Linking Reagents chemistry Mutation biology.protein Electrophoresis Polyacrylamide Gel Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences. 100:9250-9255 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1133325100 |
| Popis: | 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 ( Eco SSB) 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 Eco SSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling. Eco SSB binds to the template and to the nascent transcript and prevents the formation of a transcriptionally inert RNA:DNA hybrid. Using C-terminally truncated Eco SSB mutant proteins, human mitochondrial SSB ( Hs mt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hs mt- Eco SSB chimera, we have mapped a determinant of template recycling to the C-terminal amino acids of Eco SSB. 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 Eco SSB: It functionally substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding. |
| Databáze: | OpenAIRE |
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