MASS SPECTRAL ANALYSIS OF TRANSCRIPTION COMPLEXES

这项研究的目的是更好地了解RNA聚合酶II（RNApo II）参与转录的因素，并阐明这一过程的机制。RNA pol II的转录是蛋白质编码基因表达的重要步骤。在体外，可以用通用转录因子和RNA pol II重建准确的转录起始。然而，在体内，该过程显然更加复杂，因为转录机制必须与调控面相互作用以及与染色质模板竞争。为了更全面地了解转录机制，我将使用生物化学，遗传学，分子生物学和质谱分析，以了解在转录周期的不同阶段与RNA pol II转录复合物相关的因素。我已经开发了一个固定化模板系统，我可以快速分离功能转录复合物从酵母核提取物。洗涤复合物后，用胰蛋白酶消化相关蛋白质，并使用微柱液相色谱电喷雾电离质谱法（LC/MS/MS）进行分析。将通过使用Sequest（TM）算法搜索酿酒酵母基因组序列数据库来鉴定蛋白质。为了鉴定转录起始后保留在启动子处的组分，将在添加核苷酸三磷酸后对复合物进行类似的分析。此外，将使用质谱技术鉴定转录因子的共价修饰，如磷酸化和乙酰化。将研究相对于转录周期的修饰的时间。在确定新的因素和修改，这些因素和修改的生物相关性将通过使用分子仿制药和生物化学进行研究。基因敲除和点突变将在S.酿酒酵母，以确定因子或修饰位点对细胞生长的影响。将从在鉴定的因子中携带条件突变的菌株制备核提取物，以研究该因子在体外转录系统中的作用。这种研究转录复合物的新方法应该对转录中涉及的因素和这一过程的机制产生新的见解。由于在转录水平上调控基因表达是癌症生物学中的一个中心过程，这些研究将为我们理解这一过程做出重大贡献。

The goals of the proposed research are to gain a better understanding of the factors involved in transcription by RNA Polymerase II (RNApo II), and to elucidate the mechanisms underlying this process. Transcription by RNA pol II is an essential step in expression of protein coding genes. In vitro, accurate initiation of transcription can be reconstituted with the general transcription factors and RNA pol II. However, in vivo the process is clearly more complex as the transcription machinery must interact with regulatory faces as well as contend with the chromatin template. To obtain a more complete understanding of the transcription machinery, I will use biochemistry, genetics, molecular biology and mass spectrometry, to idea the factors associated with RNA pol II transcription complexes at different stages of the transcription cycle. I have developed an immobilized template system with which I can rapidly isolate functional transcription complexes from yeast nuclear extracts. After washing the complexes, the associated proteins will be digested with trypsin and analyzed using microcolumn liquid chromatography electrospray ionization mass spectrometry(LC/MS/MS). Proteins will be identified by searching the Saccharomyces cerevisiae genomic sequence database with the Sequest(TM) algorithm. To identify the components that remain at the promoter after initiation of transcription, a similar analysis will be performed on the complexes after addition of nucleotide triphosphates. In addition, covalent modifications of transcription factors, such as phosphorylation and acetylation, will be identified using mass spectral techniques. The timing of the modifications with respect to the transcription cycle will be investigated. Upon identifying novel factors and modifications, the biological relevance of these factors and modifications will be investigated by using molecular generics and biochemistry. Gene knockouts and point mutations will be engineered in S. cerevisiae, to determine the effect of the factor or modification site on cell growth. Nuclear extracts will be prepared from strains carrying conditional mutations in the identified factor to study the role of the factor in in vitro transcription systems. This novel approach to study transcription complexes should yield new insights into the factors involved in transcription and the mechanisms underlying this process. Since regulation of gene expression at the level of transcription is a central process in cancer biology, these studies will make significant contributions to our understanding of this process.