Transcriptional repressor CopR acts by inhibiting RNA polymerase binding.
CopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in Bacillus subtilis. It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential repR mRNA about 10- to 20-fold. In previous work, DNA binding and dimerization constants were determined and the motifs responsible localized. The C terminus of CopR was shown to be required for stability. Furthermore, SELEX of the copR operator revealed that in vivo evolution was for maximal binding affinity. Here, we elucidate the repression mechanism of CopR. Competition assays showed that CopR-operator complexes are 18-fold less stable than RNA polymerase (RNAP)-pII complexes. DNase I footprinting revealed that the binding sites for CopR and RNAP overlap. Gel-shift assays demonstrated that CopR and B. subtilis RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO(4) footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.
Friedrich-Schiller-Universität Jena, Biologisch-Pharmazeutische Fakultät, AG Bakteriengenetik, Philosophenweg 12, Jena D-07743, Germany.
Andreas
A
Licht
Friedrich-Schiller-Universität Jena, Biologisch-Pharmazeutische Fakultät, AG Bakteriengenetik, Philosophenweg 12, Jena D-07743, Germany.
Peggy
P
Freede
Friedrich-Schiller-Universität Jena, Biologisch-Pharmazeutische Fakultät, AG Bakteriengenetik, Philosophenweg 12, Jena D-07743, Germany.
Sabine
S
Brantl
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D018983:DNA Footprinting; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D008954:Models, Biological; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*
D004269:DNA, Bacterial; D012329:RNA, Bacterial; C096035:RNAIII, Staphylococcus aureus; D012097:Repressor Proteins; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I