Evidence that Bacillus catabolite control protein CcpA interacts with RNA polymerase to inhibit transcription

Bacillus catabolite control protein (CcpA) mediates carbon catabolite repression (CCR) by controlling expression of catabolite responsive (CR) genes or operons through interaction with catabolite responsive elements (cres) located within or outside of CR promoters. Here, we investigated how CcpA inhibits the transcription of CR promoters in vitro. CcpA has different affinities for different cres, but this does not correlate with its ability to inhibit transcription. In the amyE promoter, which overlaps a CcpA binding site (amyE cre centred at +4.5), CcpA does not prevent RNA polymerase (RNAP) binding to the promoter; it may even interact with RNAP. Inserting non-integral turns of helix (1.5 and 2.5) between the amyE promoter (-10 hexamer) and the amyE cre relieved CCR of amyE expression. In the xyl operon, despite the downstream location of its cre (a major cre centred at +130.5), CcpA blocked transcription initiation, not elongation (roadblock) at the site of the cre. Taken together, our results strongly suggest that CcpA requires interactions with RNAP to inhibit transcription.