Formation of Intermediate Transcription Initiation Complexes at p fliD and p flgM by ς 28 RNA Polymerase

Abstract

ABSTRACT 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 p fliC 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 p fliC , p fliD , and p flgM . However, certain aspects of DNA strand separation and complex stability are promoter dependent. Open complexes form in a concerted manner at p flgM , while a sequential pattern of open complex formation occurs at p fliD . Open and initiated complexes formed by holoenzyme carrying ς 28 are generally unstable to heparin challenge, with the exception of initiated complexes at p flgM , which are stable in the presence of nucleoside triphosphates.