title: Derepression of bacterial transcription-repair coupling factor is associated with a profound conformational change.
abstract: Transcription-repair coupling factor (TRCF; the product of the mfd gene) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNA polymerase stalled at a noncoding lesion in the DNA template strand, uses the energy from ATP hydrolysis to disrupt the transcription complex, and stimulates DNA repair by recruiting UvrA, a component of the nucleotide excision repair machinery, to the site. TRCF is a large (130 kDa) multifunctional protein with a complex structure-function relationship consisting of a compact arrangement of eight structured domains linked by flexible linkers. Through a conserved, intramolecular, interdomain interaction, TRCF is held in a conformation in which its enzymatic activities (ATPase activity and DNA translocase activity) are strongly repressed. Disruption of the repressive interdomain interaction by amino acid substitutions within the interface derepresses ATPase and DNA translocase activities. In this work, we have shown that derepressed TRCF mutants are dramatically sensitized to limited proteolysis compared with repressed TRCF, pointing to an altered conformational state. Analysis of the protease cleavage sites mapped onto the structure of the repressed TRCF conformation indicates that (1) the cleavage sites tend to cluster at linkers connecting the TRCF structured domains, and (2) many of the cleavage sites sensitized in the derepressed TRCF are partially or completely buried to protease access in the repressed TRCF structure. We conclude that TRCF derepression is associated with profound conformational changes that primarily involve a reorganization of the interdomain interactions.
authors.affiliation: The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Devendra B
authors.initials: DB
authors.lastname: Srivastava
authors.forename: Seth A
authors.initials: SA
authors.lastname: Darst
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004260:DNA Repair; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C072103:transcription repair coupling factor protein, Bacteria