A Pyrococcus homolog of the leucine-responsive regulatory protein, LrpA, inhibits transcription by abrogating RNA polymerase recruitment.
The genomes of Archaea harbor homologs of the global bacterial regulator leucine-responsive regulatory protein (Lrp). Archaeal Lrp homologs are helix-turn-helix DNA-binding proteins that specifically repress the transcription of their own genes in vitro. Here, we analyze the interaction of Pyrococcus LrpA with components of the archaeal transcriptional machinery at the lrpA promoter. DNA-protein complexes can be isolated by electrophoretic mobility shift assays that contain both LrpA and the two archaeal transcription factors TBP and TFB. Phenanthroline-copper footprinting experiments showed that the DNA-binding sites of LrpA and TBP/TFB do not overlap. These results and the finding that association of RNA polymerase with the TBP-TFB promoter complex was inhibited in the presence of LrpA indicate that Pyrococcus LrpA interferes with RNA polymerase recruitment. A DNA motif required for the LrpA-DNA interaction was inferred from dimethylsulfate methylation interference experiments.
Dahlke
Isabell
I
Institut für Allgemeine Mikrobiologie, Universität Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
Thomm
Michael
M
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D051756:Leucine-Responsive Regulatory Protein; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics; D013463:Sulfuric Acid Esters / Q000378:metabolism; D035181:TATA-Box Binding Protein; D013698:Templates, Genetic; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; C418107:LrpA protein, Pyrococcus furiosus; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010618:Phenanthrolines; D013463:Sulfuric Acid Esters; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; C106466:transcription factor B, Archaea; D051756:Leucine-Responsive Regulatory Protein; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C007482:dimethyl sulfate; C025205:1,10-phenanthroline