PrfA mediates specific binding of RNA polymerase of Listeria monocytogenesto PrfA-dependent virulence gene promoters resulting in a transcriptionally active complex

There is accumulating evidence that the coordinate transcription of the vir ulence genes in Listeria monocytogenes constitutes a very complex regulatio n mechanism which might require other factors in addition to PrfA. We previ ously described an unknown proteinaceous component from crude bacterial cel l extracts, which, together with PrfA, formed a specific complex (CI) in el ectrophoretic mobility shift assays (EMSA) with an hly promoter probe. Here we identify the RNA polymerase (RNAP) of L. monocytogenes as an essential component of the CI complex. Addition of purified RNAP plus PrfA to the hly promoter probe allowed reconstitution of a complex migrating at the same h eight as CI. By using EMSA and DNaseI footprint experiments it could be sho wn that PrfA leads to an enhanced and specific binding of RNAP. Transcripti onal activity of RNAP in vitro, using the actA promoter, was strictly depen dent on PrfA.