This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 'Quantitative mass spectrometry was used to comprehensively characterize a yeast RNA polymerase II (pol II) preinitiation complex (PIC). Complexes were isolated from nuclear extracts by using immobilized promoter DNA templates. The nuclear extract was derived from a strain carrying a temperature-sensitive allele of the TATA binding protein (TBP). This extract is defective in PIC formation, but addition of recombinant TBP to the reaction restores PIC formation. Quantitative mass spectrometry was performed on proteins isolated from PIC formation assays in the presence and absence of recombinant TBP. Of 68 known components of the PIC, 59 were identified. In addition, an uncharacterized protein was found. Extracts prepared from a deletion strain show a severe defect in transcription assays. It is likely that this is a new component of the pol II PIC. The Aebersold laboratory would like to initiate a collaboration to identify proteins that interact with this new factor by two-hybrid analysis.
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