CHARACTERIZATION OF A NOVEL PROTEIN THAT INTERACTS WITH THE RNA POLYMERASE II P

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。定量质谱法用于全面表征酵母RNA聚合酶II（pol II）前起始复合物（PIC）。通过使用固定化启动子DNA模板从核提取物中分离复合物。核提取物来自携带TATA结合蛋白（TBP）的温度敏感等位基因的菌株。该提取物在PIC形成中是有缺陷的，但向反应中加入重组TBP可恢复PIC形成。在存在和不存在重组TBP的情况下，对从PIC形成测定中分离的蛋白质进行定量质谱分析。在事先知情同意的68种已知成分中，确定了59种。此外，还发现了一种未知的蛋白质，从缺失菌株中提取的蛋白质在转录检测中表现出严重的缺陷。这可能是pol II PIC的一个新组件。Aebersold实验室希望发起一项合作，通过双杂交分析鉴定与这种新因子相互作用的蛋白质。

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.