MASS SPECTRAL ANALYSIS OF TRANSCRIPTION COMPLEXES

这项研究的目的是为了更好地了解RNA聚合酶II(RNApo II)转录过程中涉及的因素，并阐明这一过程的机制。RNA polII的转录是蛋白质编码基因表达的重要步骤。在体外，一般转录因子和RNA polII可以准确地重组转录起始。然而，在体内，这个过程显然更加复杂，因为转录机制必须与调控面相互作用，并与染色质模板竞争。为了更完整地了解转录机制，我将利用生物化学、遗传学、分子生物学和质谱学，来构思与RNA PolII转录复合体相关的因素在转录周期的不同阶段。我已经开发了一种固定化模板系统，利用它我可以快速从酵母核提取物中分离出功能转录复合体。洗涤后，结合蛋白用胰酶消化，用微柱液相色谱电喷雾电离质谱仪(LC/MS/MS)分析。蛋白质将通过SEQUEST(TM)算法搜索酿酒酵母基因组序列数据库来鉴定。为了确定转录开始后仍保留在启动子上的成分，在添加三磷酸核苷酸后，将对复合体进行类似的分析。此外，转录因子的共价修饰，如磷酸化和乙酰化，将使用质谱学技术进行鉴定。将研究相对于转录周期的修改的时间。在确定新的因子和修饰后，将使用分子遗传学和生物化学来研究这些因子和修饰的生物学相关性。基因敲除和点突变将在酿酒酵母中进行工程，以确定因子或修饰位置对细胞生长的影响。将从携带已识别因子的条件突变的菌株中制备核提取液，以研究该因子在体外转录系统中的作用。这种研究转录复合体的新方法应该会对转录过程中涉及的因素和这一过程背后的机制产生新的见解。由于转录水平的基因表达调控是癌症生物学的中心过程，这些研究将对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.