VIRAL REGULATION OF RIBOSOMAL RNA TRANSCRIPTION

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DESCRIPTION (adapted from the applicant's abstract): Simian virus 40 (SV40) large T antigen is a multifunctional regulatory protein that plays a key role in the viral life cycle. In addition, large T antigen immortalizes primary cells, and induces cell transformation and tumor formation in animals. To accomplish these functions, large T antigen has to alter the cellular mechanisms that control the expression of genes involved in cell cycle progression and cell proliferation. Ribosomal RNA synthesis by RNA polymerase I (pol I) is tightly associated with cell growth and proliferation, and previous studies demonstrated that large T antigen up-regulates RNA pol I transcription in SV40-infected cells. To better understand how large T antigen stimulates RNA pol I transcription, he has established an in vitro transcription system that responds to large T antigen. During these studies, his laboratory has discovered that the transcription factor SL1 is one of the cellular components of the pol I transcriptional machinery targeted by large T antigen. In addition, these studies suggested that, in part, the phosphorylation of UBF by a still unidentified T antigen-associated kinase, may also contribute to the overall stimulatory process. In this grant proposal, using a biochemical approach, the PI proposed to dissect the mechanism of regulation by RNA pol I transcription by large T antigen. The first goal is to understand the role of large T antigen-SL1 interaction in the stimulatory process. For this purpose, he will analyze the effects of deletion and single point mutations in large T antigen in a variety of in vivo and in vitro protein-protein interaction assays and trancription assays, and correlate the ability to bind to SL1 with the transcriptional stimulation. Second, he proposes to use footprinting and EMSA assays to test whether these protein-protein interactions may potentiate functional interactions between the transcription factors and facilitate the formation of the transcription initiation complex. Interestingly, his preliminary studies indicate that the region of large T antigen necessary for pol I stimulation overlaps with the domain involved in the binding and inactivation of p53. Thus, he has designed a set of experiments to determine the potential functional link between the tumor suppressor p53 and large T antigen stimulation of pol I transcription. Finally, he will address the significance and the role of UBF phosphorylation in the T antigen-mediated activation process and characterize the associated-protein kinase activity. These studies will provide new insights on the molecular mechanisms of mammalian gene regulation and will unravel some of the strategies adopted by small DNA viruses such as SV40 to alter the regulation of key cellular processes.