Date of Award
Doctor of Philosophy (PhD)
Transcription, the process of copying information encoded in DNA into RNA, to facilitate the expression of encoded proteins, is a central process in all living organisms. The expression and repression of subsets of genes allows different cell types in an organism to maintain diverse physiological roles and permits individual cells to respond to various environmental stimuli. Transcription in prokaryotic cells is performed by a single macromolecular complex, RNA polymerase. In rapidly growing cells with abundant resources, prokaryotic RNA polymerase is mostly located at ribosomal RNA (rRNA) promoters, actively transcribing the large, structured RNAs required for protein translation. As resources become more scarce, RNA polymerase directly responds to cellular signals that lead to the repression of rRNA transcription. This regulation has long been thought to be driven primarily by small molecule effectors that signal scarcity. In this thesis, I will report work done on two transcription factors in prokaryotes that regulate RNA polymerase activity at rRNA promoters. The Staphylococcus aureus (Sau) Phage G1 protein PhERI (previously ORF67), was previously described as a general RNA polymerase inhibitor. PhERI expression in Sau cells inhibits cell growth, which could have therapeutic potential against this deadly pathogen. I describe the structure of PhERI bound to Sau σ A 4, the region of RNA polymerase to which it binds. While PhERI interacts with RNAP through σ, I show that RNA polymerase activity at most -10/-35 promoters is not affected. Structural, biochemical and genomic approaches demonstrate that PhERI interacts with σA 4 near the -35 element of all promoters, but blocks the binding of an additional RNA polymerase subunit, the α-CTD, to UP-element DNA sequences. PhERI therefore only inhibits RNA polymerase activity at promoters requiring UP-element activation, most notably the rRNA promoters. This work not only delineates the mechanism of PhERI but also describes novel -10/-35 promoters in Staphlococcus aureus, defines rRNA promoters in this organism for the first time, and shows UP-element activation is required for rRNA transcription. The mycobacterial protein CarD is known to interact with RNA polymerase, but its impact on transcription directly at promoters has not been described. The structure of the Thermus Thermophilus CarD was solved in the lab, allowing a model for the interaction between CarD and RNA polymerase to be built. I show that CarD stimulates RNA polymerase activity at rRNA promoters, but not all promoters, by directly stabilizing the RNA polymerase open complex on promoter DNA. These two proteins both exploit unique parameters of RNA polymerase at rRNA promoters to specifically regulate RNA polymerase activity at these functionally important promoters.
Osmundson, Joseph S., "rRNA Promoters as Targets for Transcription Factors: Structural and Functional Studies of PhERI and CarD" (2012). Student Theses and Dissertations. 248.