Our long-term goal is to understand the structural basis for the regulation of RNA polymerase II machinery in the transcription cycle. The eukaryotic mRNA synthesis system has emerged as an enormous protein apparatus consisting of RNA polymerase II (Pol II) at its heart surrounded by layers of controlling factors. We recently cracked the phase problem for the 500 kDa 10-subunit Pol H from yeast. This advent has made it feasible to unveil the apparatus in a more elaborate fashion, so we can analyze Pol IT-factor(s) interactions that modulate the transcription activities of Pol II. Transcription is the first step in gene expression. Signals from many cellular processes such as nutrient sensing, catabolite repression, hormone stimulation, differentiation and responses to environmental stimuli ultimately reach the level of transcription to exert their effects. Aberration of transcriptional control is one of the mechanisms underlying tumorigenesis, since cancer arises from disruption of the fine balance between proliferative and differentiative genetic programs. Oncogene products are frequently found to be key players in the control networks of signal transduction, cell cycle and transcription regulations. We propose to examine, in yeast, various Pol TI-factor(s) complexes corresponding to distinct functional steps in the transcription cycle. Biochemical and X-ray crystallographic methods developed in the work of the 10-subunit Pol TI and the ribosome will be employed, refined and extended to tackle this new generation of structural problems in transcription regulation.
Specific aims for the project period include determinations of the complete structures of Pol H alone and in complex with TFIIF and Fcp1 Pol TI-TFIIF is an invariant subcomplex of a much larger transcription pre-initiation complex (PlC). It is the form of Pol II that enters into the PlC. The Pol ll-Fcp1 complex relates to steps in elongation and termination/re-initiation in the transcription cycle. FcpT, an essential gene, is a phosphatase specific for the C-terminal heptapeptide repeat domain (CTD) of the largest subunit of Pol II. Fcp1 influences Pol II activity through modulation of the dynamic balance between CTD phosphorylation and dephosphorylation. The Pol TI-factor complex structures are required for addressing the Pol II regulation issues.
