Abstract
CcpN, a transcriptional repressor from Bacillus subtilis that is responsible for the carbon catabolite repression of three genes, has been characterized in detail in the past 4 years. However, nothing is known about the actual repression mechanism as yet. Here, we present a detailed study on how CcpN exerts its repression effect at its three known target promoters of the genes sr1, pckA, and gapB. Using gel shift assays under non-repressive and repressive conditions, we showed that CcpN and RNA polymerase can bind simultaneously and that CcpN does not prevent RNA polymerase (RNAP) binding to the promoter. Furthermore, we investigated the effect of CcpN on open complex formation and demonstrate that CcpN also does not act at this step of transcription initiation at the sr1 and pckA and presumably at the gapB promoter. Investigation of abortive transcript synthesis revealed that CcpN acts differently at the three promoters: At the sr1 and pckA promoter, promoter clearance is impeded by CcpN, whereas synthesis of abortive transcripts is repressed at the gapB promoter. Eventually, we demonstrated with Far Western blots and co-elution experiments that CcpN is able to interact with the RNAP alpha-subunit, which completes the picture of the requirements for the repressive action of CcpN. On the basis of the presented results, we propose a new working model for CcpN action.