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Volume 157, Issue 4

Transcriptional repressor CopR acts by inhibiting RNA polymerase binding Free

Abstract

CopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential 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 operator revealed that 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 RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.

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Keyword(s): EMSA, electrophoretic mobility shift assay , PAA, polyacrylamide , RNAP, RNA polymerase , SELEX, systematic evolution of ligands by exponential enrichment (in vitro selection) and αCTD, C-terminal domain of the α subunit of RNA polymerase
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2011-04-01
2024-04-24
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Transcriptional repressor CopR acts by inhibiting RNA polymerase binding
Microbiology 157, 1000 (2011); https://doi.org/10.1099/mic.0.047209-0
/content/journal/micro/10.1099/mic.0.047209-0
/content/journal/micro/10.1099/mic.0.047209-0
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