Our long-term goal is to understand the structural basis for the regulation of RNA polymerase II Imachinery 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 10-subunit Pol II (500 kDa) from yeast. This advent has made it feasible to unveil the apparatus in a more elaborate fashion, so we can analyze Pol II-factor(s) interactions that modulate the transcription activities of Pol I1. 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 II-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 II 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 are crystal structure determinations of three Pol II-factor complexes, including the pre-mRNA 5' capping enzyme, a Pol II specific phosphatase Fcpl and a subassembly of the Pol II transcription preinitiation complex. ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Tompkins, Laurie
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Medical College of Wisconsin
Schools of Medicine
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