Formation of Intermediate Transcription Initiation Complexes at pfliD and pflgM by ς(28) RNA Polymerase

The ς subunit of prokaryotic RNA polymerase is an important factor in the control of transcription initiation. Primary ς factors are essential for growth, while alternative ς factors are activated in response to various stimuli. Expression of class 3 genes during flagellum biosynthesis in Salmonella enterica serovar Typhimurium is dependent on the alternative ς factor ς(28). Previously, a novel mechanism of transcription initiation at the fliC promoter by ς(28) holoenzyme was proposed. Here, we have characterized the mechanism of transcription initiation by a holoenzyme carrying ς(28) at the fliD and flgM promoters to determine if the mechanism of initiation observed at pfliC is a general phenomenon for all ς(28)-dependent promoters. Temperature-dependent footprinting demonstrated that promoter binding properties and low-temperature open complex formation are similar for pfliC, pfliD, and pflgM. However, certain aspects of DNA strand separation and complex stability are promoter dependent. Open complexes form in a concerted manner at pflgM, while a sequential pattern of open complex formation occurs at pfliD. Open and initiated complexes formed by holoenzyme carrying ς(28) are generally unstable to heparin challenge, with the exception of initiated complexes at pflgM, which are stable in the presence of nucleoside triphosphates.