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Jennifer Givens

Advisor: Alicia Dombroski, Ph.D.

The dissociable sigma subunit of prokaryotic RNA polymerase is an important factor in the control of transcription initiation. In order to initiate transcription, RNA polymerase must associate with a sigma factor. Promoter specificity is determined by the particular sigma factor associated with RNA polymerase and thus the use of various sigma factors allows bacteria to maintain basal gene expression and to regulate gene expression in response to environmental or developmental signals. Primary sigma factors are essential for growth, while alternative sigma factors are activated in response to various stimuli. Expression of class three genes during flagellum biosynthesis in Salmonella enterica serovar Typhimurium is dependent on the alternative sigma factor, sigma28. Sigma28 is unusual because it lacks an amino terminal region, region 1.2, which is commonly found in other sigma factors. Previously, a novel mechanism of transcription initiation at the fliC promoter by sigma28 holoenzyme was proposed. Here, the mechanism of transcription initiation by holoenzyme carrying sigma28 at the fliD and flgM promoters was characterized to determine if the mechanism of initiation observed at pfliC is a general phenomenon for all sigma28-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 sigma28 are generally unstable to heparin challenge, with the exception of initiated complexes at pflgM, which are stable in the presence of NTPs.

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Research Info

Formation of Intermediate Transcription Initiation Complexes at pfliD and pflgM by Sigma28 RNA Polymerase