In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element.
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.
Bailey
M J
MJ
Department of Pathology, University of Cambridge, UK.
Hughes
C
C
Koronakis
V
V
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D009876:Operon; D010445:Peptide Elongation Factors / Q000235:genetics* / Q000378:metabolism*; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; C106401:RfaH protein, E coli; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases