Transcription initiation at the flagellin promoter by RNA polymerase carrying sigma(28) from Salmonella typhimurium

The sigma subunit of RNA polymerase is a critical factor in positive contro l of transcription initiation. Primary a factors are essential proteins req uired for vegetative growth, whereas alternative sigma factors mediate tran scription in response to various stimuli. Late gene expression during flage llum biosynthesis in Salmonella typhimurium is dependent upon an alternativ e a factor, sigma(28), the product of the fliA gene. We have characterized the intermediate complexes formed by sigma(28) holoenzyme on the pathway to open complex formation. Interactions with the promoter for the flagellin g ene fliC were analyzed using DNase I and KMnO4 footprinting over a range of temperatures. We propose a model in which closed complexes are established in the upstream region of the promoter, including the -35 element, but wit h little significant contact in the -10 element or downstream regions of th e promoter. An isomerization event extends the DNA contacts into the -10 el ement and the start site, with loss of the most distal upstream contacts ac companied by DNA melting to form open complexes, Melting occurs efficiently even at 16 degrees C. Once open complexes have formed, they are unstable t o heparin challenge even in the presence of nucleoside triphosphates, which have been observed to stabilize open complexes at rRNA promoters.