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Olivia Lee Schaubach

Advisor: Alicia Dombroski, Ph.D.

Prokaryotic RNA polymerase is a multimeric enzyme with a molecular composition of a2bbÕs, that catalyzes RNA synthesis. The dissociable σ subunit of RNA polymerase is a critical factor in the positive control of transcription initiation. Promoter specificity is determined by the particular s factor associated with polymerase, thus regulating which genes will be transcribed. Primary s factors are essential proteins required for vegetative growth, whereas alternative s factors mediate transcription in response to various stimuli. Late gene expression during flagellum biosynthesis in Salmonella typhimurium is dependent upon an alternative s factor, s28, the product of the fliA gene. s28 is an unusual a factor because it lacks an amino terminal region, region 1.2, that is commonly found in other s factors, implying a potential variation in function. The intermediate complexes formed by s28 holoenzyme were characterized on the pathway to open complex formation. Interactions with the AT-rich upstream region including the promoter for the flagellin gene, fliC, were analyzed using DNase I and KMnO4 footprinting over a range of temperatures. Here, a novel mechanism has been proposed in which closed complexes are established in the upstream region of the promoter, including the -35 element, but with little significant contact in the -10 element or downstream regions of the promoter. An isomerization event extends the DNA contacts into the -10 element and the start site, with loss of the most distal upstream contacts accompanied by DNA melting to form open complexes. Melting occurs efficiently even at 16¡C. Once open complexes have formed, they are unstable to heparin challenge even in the presence of nucleoside triphosphates, which have been observed to stabilize open complexes at ribosomal RNA promoters. The variations in amino termini of s factors appear to effect the mechanism of RNA polymerase transcription initiation. A chimeric s factor was constructed containing the amino terminal portion of s70, region 1 and spacer, and s28 to examine mechanistic differences that are dependent on the s factor structure.

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Research Info

Mechanism of Transcription Initiation by s28 RNA