Regulatory sequences in sigma 54 localise near the start of DNA melting

Transcription initiation by the enhancer-dependent sigma (54) RNA polymeras e holoenzyme is positively regulated after promoter binding. The promoter D NA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron che late probes attached to amino and carboxyl-terminal domains of sigma (54) w ere used to map sigma (54)-DNA interaction sites. The two domains localise to form a centre over the -12 promoter region. The use of deletion mutants of sigma (54) suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationsh ip between the centre and promoter DNA changes. We suggest that the activat or re-organises the centre to favour stable open complex formation through adjustments in sigma (54)-DNA contact and sigma (54) conformation. The cent re is close to the active site of the RNA polymerase and includes sigma (54 ) regulatory sequences needed for DNA melting upon activation. This contras ts systems where activators recruit RNA polymer ase to promoter DNA, and th e protein and DNA determinants required for activation localise away from p romoter sequences closely associated with the start of DNA melting. (C) 200 1 Academic Press.