(adapted in part from applicant's description): This project examines the mechanism of action of the TFIIS (SII) transcript cleavage factor and its role in transcriptional regulation. SII stimulates transcription by binding to paused or arrested RNA polymerase II and activating a latent nuclease in the enzyme that cleaves the nascent transcript near its 3' end to enable readthrough of various blocks to chain elongation. This project will continue to explore SII's mechanism with a focus on its interaction with nascent RNA. The contact site of RNA on SII will be precisely mapped. Mutant SII proteins eliminating RNA contact will be generated and tested for in vitro activity and an ability to complement the phenotypes of an SII gene deletion in yeast. SII contact sites on the polymerase will be mapped by protease footprinting. Direct evidence for arrested RNA polymerase II on chromatin in vivo will be sought using nuclear run-on and chromatin immunoprecipitation assays with anti-polymerase antibodies. Genes with biased base content will be surveyed to see if NTP pools regulate elongation efficiency in a gene-specific manner in vivo. Another aim is to determine the mechanism used to regulate PUR5 expression in response to NTP pool reduction in yeast. This will include measurement of NTP pools under various growth conditions and in response to 6-azauracil, an inhibitor of UTP and GTP synthesis. Finally, mice disrupted for two SII family genes (TceaI and Tcea3) will be generated and analyzed for developmental phenotypes.
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