YEAST GENES INVOLVED IN GENERAL TRANSCRIPTIONAL CONTROL

参与一般转录控制的酵母基因

• 批准号: 2770958
• 负责人: CLYDE L DENIS
• 金额: $ 32.79万
• 依托单位: UNIVERSITY OF NEW HAMPSHIRE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1999-09-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2770958
• 关键词: DNA directed RNA polymerase; affinity chromatography; alcohol dehydrogenase; cell cycle proteins; cell growth regulation; chemical binding; chimeric proteins; fungal genetics; gel filtration chromatography; gene deletion mutation; genetic transcription; immunoprecipitation; intermolecular interaction; molecular cloning; molecular size; northern blottings; phosphorylation; protein kinase; protein purification; protein reconstitution; protein sequence; protein structure function; suppressor mutations; transcription factor; western blottings

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.

这项研究的目的是阐明桉树的机制 基因调节。选择进行研究的系统是酵母调节 由CCR4，CAF1，CAF185和其他蛋白质组成的复合物 正确表达许多不同基因的基因所必需 酵母。至少包括CAF1和CAF185在内的五种蛋白质已经 被识别为与CCR4的共免疫沉淀，另外七个出现 与之共纯化。在较高的 生物和哺乳动物CAF1可以代替几种酵母CAF1 功能。生化证据表明CCR4和CAF1可以是 专门保留在RNA聚合酶H C末端结构域（CTD）上 亲和力柱，表明CCR4/CAF1可能是外围组件 核心转录设备。 CCR4蛋白复合物是 与其他几种调节络合物不同 在包括SNF/SWI蛋白的酵母中鉴定 Holoenzyme SRB复合物，TATA结合蛋白相关的因素和 SPT6复合物。该赠款提案的主要重点是两个方面： 1）识别和表征CCR4蛋白复合物的成分 2）确定该复合物如何与转录相互作用 影响转录的机械。 CCR4复合物的组件将通过净化来识别 复杂到同质性。单个组件的基因将是 从每种蛋白质的肽片段进行微链测序后克隆。 配合物中的组装和蛋白质相互作用将是 通过遗传和生化技术分析。 CCR4和 它相关的蛋白质将通过确定其作用来详细阐述 在体外转录上，通过识别其与 转录机械，并检查其与RNA CTD的链接 聚合酶II。 CCR4与DBF2的关系，DBF2是较晚的有丝分裂 细胞周期调节的蛋白激酶，与 CCR4也将进行研究。最后，其他调节蛋白 与CCR4功能相关的功能将通过表征和 克隆在突变后导致合成致死性的基因 结合CCR4删除。