In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element
| Autor: | Colin Hughes, Vassilis Koronakis, Marc J. A. Bailey |
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| Rok vydání: | 2000 |
| Předmět: |
Transcription
Genetic RNA polymerase II RNA polymerase complex Biology Ribosome chemistry.chemical_compound Adenosine Triphosphate Cytosol Bacterial Proteins Transcription (biology) RNA polymerase Operon Escherichia coli Genetics Molecular Biology General transcription factor Escherichia coli Proteins Hydrolysis DNA-Directed RNA Polymerases Peptide Elongation Factors RNA Bacterial chemistry Biochemistry Transcription preinitiation complex Trans-Activators biology.protein Transcription factor II D Ribosomes |
| Zdroj: | Molecular and General Genetics MGG. 262:1052-1059 |
| ISSN: | 1432-1874 0026-8925 |
| DOI: | 10.1007/pl00008648 |
| Popis: | An unusual regulatory mechanism that controls transcription elongation in long fertility and virulence operons in bacteria is effected by two specialised components, the RfaH protein and the nucleic acid ops element. Without direct interaction, ops acts to reduce the concentration of RfaH required to stimulate distal gene transcription, and we have proposed that ops recruits RfaH to the transcription machinery. To provide direct experimental evidence for this view, we used gel fitration to identify potential RfaH complexes assembled in Escherichia coli cell extracts that carry out RfaH-dependent transcription. This novel molecular weight shift assay revealed that RfaH-dependent transcription elongation occurs concomitantly with recruitment of RfaH into a high molecular weight transcription complex, and that this recruitment is specifically directed by the ops element. Assembly of this complex required RNA polymerase and nucleotide hydrolysis, but not processive transcription. Neither assembly of the complex nor RfaH-dependent transcription was observed in in vitro reactions containing only ops, RfaH and purified core (alphabetabeta') RNA polymerase; both processes required the combination of subcellular fractions containing the RNA polymerase complex, the cytoplasmic membrane and ribosomes. The data confirm that the ops element directs recruitment of RfaH into a multi-component RNA polymerase complex that resists transcription termination. |
| Databáze: | OpenAIRE |
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