YEAST GENES INVOLVED IN GENERAL TRANSCRIPTIONAL CONTROL

本研究的目的是阐明真核生物的作用机制， 基因调控选择的研究系统是酵母调节 由CCR 4、CAF 1、CAF 185和其他蛋白质组成的复合物， 需要适当表达的多种基因， 酵母至少有五种蛋白质，包括CAF 1和CAF 185， 被鉴定为与CCR 4共免疫沉淀， CAF 1的同源物已经在高等植物中鉴定， 哺乳动物CAF 1可以替代几种酵母CAF 1 功能协调发展的生物化学证据表明，CCR 4和CAF 1可以被 特异性保留在RNA聚合酶H C-末端结构域（CTD）上 CCR 4/CAF 1可能是细胞的外周成分 核心转录器的一部分CCR 4蛋白复合物是 与其他几种调控复合体不同， 包括SNF/SWI蛋白， 全酶SRB复合物，TATA结合蛋白相关因子，和 SPT 6复合体这项赠款提案的中心重点是两方面的： 1)鉴定和表征CCR 4蛋白复合物的组分 和2）确定该复合物如何与转录因子相互作用， 机器影响转录。 CCR 4复合物的组分将通过纯化 复杂到同质。单个组分的基因将 在对来自每种蛋白质的肽片段进行微测序后克隆。 复合物内的组装和蛋白质相互作用将是 通过基因和生化技术进行分析。CCR 4的功能和 它的相关蛋白质将通过确定它们的作用来阐述， 在体外转录，通过确定他们的接触， 转录机制，并通过检查它们的链接到CTD的RNA 聚合酶II。CCR 4与晚期有丝分裂抑制因子DBF 2的关系 细胞周期调节蛋白激酶，与 CCR 4也将被研究。最后，另外的调节蛋白， 与CCR 4功能性相关将通过表征和 克隆基因，当突变时， 与CCR 4缺失的组合。

The goal of this research is to elucidate the mechanisms of eucaryotic gene regulation. The system chosen for study is the yeast regulatory complex consisting of CCR4, CAF1, CAF185 and other proteins which are required for the proper expression of a number of diverse genes in yeast. At least five proteins, including CAF1 and CAF185, have been identified as co-immuneprecipitating with CCR4 and another seven appear to co-purify with it. Homologs to CAF1 have been identified in higher organisms, and mammalian CAF1 can substitute for several of yeast CAF1 functions. Biochemical evidence indicates that CCR4 and CAF1 can be specifically retained on a RNA polymerase H C-terminal domain (CTD) affinity column, suggesting that CCR4/CAF1 may be peripheral components of the core transcription apparatus. The CCR4 protein complex is distinct from several other regulatory complexes that have been identified in yeast including that of the SNF/SWI proteins, the holoenzyme SRB complex, the TATA-binding protein associated factors, and the SPT6 complex. The central focus of this grant proposal is two-fold: 1) Identify and characterize the components of the CCR4 protein complex and 2) determine how this complex interacts with the transcriptional machinery to affect transcription. The components of the CCR4 complex will be identified by purifying the complex to homogeneity. The genes for individual components will be cloned following microsequencing of peptide fragments from each protein. The assembly and protein interactions within the complex will be analyzed by genetic and biochemical techniques. The function of CCR4 and its associated proteins will be elaborated by determining their effects on in vitro transcription, by identifying their contacts with the transcriptional machinery, and by examining their link to the CTD of RNA polymerase II. The relationship of CCR4 to that of DBF2, a late mitotic cell-cycle regulated protein kinase that associates genetically with CCR4, will also be studied. Finally, additional regulatory proteins that functionally relate with CCR4 will be identified by characterizing and cloning the genes which when mutated result in synthetic lethality in combination with a ccr4 deletion.