YEAST GENES INVOLVED IN GENERAL TRANSCRIPTIONAL CONTROL

参与一般转录控制的酵母基因

• 批准号: 6180117
• 负责人: CLYDE L DENIS
• 金额: $ 36.6万
• 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 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180117
• 关键词: cell cycle; fungal genetics; gene expression; genetic models; genetic regulation; genetic regulatory element; genetic transcription; mass spectrometry; protein protein interaction; protein purification; protein structure function; transcription factor

The goal of this research is to elucidate the mechanisms of eucaryotic gene regulation. The system chosen to study is the multi-protein CCR4-NOT complex, which affects gene expression both positively and negatively. It consists of two complexes, 1.9 and 1.0 mD in size, that are distinct from other transcriptionally important groups of proteins, such as the SNF/SWI and SAGA complexes, TFIID, and the RNA polymerase II holoenzyme. The smaller of the CCR4-NOT complexes contains CCR4, CAF1, the five NOT proteins and several unidentified proteins. The repressive effects of the CCR4-NOT proteins appear to occur through destabilizing TBP association with non-canonical TATAA sequences, probably through their direct interaction with TFIIA, TAFs or other TBP-associated factors. The SRB9-11 holoenzyme subcomplex also displays physical and functional interactions with the CCR4-NOT complex. The central focus of this grant proposal is three-fold: 1) What are the components and physiological roles of the two CCR4-NOT complexes? 2) How do the CCR4-NOT proteins affect the function of TBP and its associated factors? and 3) How is the CCR4-NOT complex required for activated transcription? The 1.9 mD CCR4-NOT complex will be isolated, its components cloned following mass spectrometric analysis, and their functions characterized. The mechanism by which the CCR4-NOT complex affects the action of TBP and its associated factors will be elaborated by several biochemical studies. Genetic screens will be undertaken to identify new factors or novel interactions between the CCR4-NOT complex and transcriptionally important proteins. CCR4 activator function will be explored through characterizing its unique C- and N-terminal interactions with SRB9-11, SPT5, TAF60, and other proteins. The key features of the ADH2 promoter which are responsive to the CCR4-NOT complex will be identified to determine how the CCR4-NOT complex controls the ADH2 gene in a positive manner.

本研究的目的是阐明真核生物基因调控的机制。选择研究的系统是多蛋白CCR4-NOT复合物，它对基因表达有正向和负向影响。它由1.9和1.0 mD大小的两个复合物组成，不同于其他转录重要的蛋白质群，如SNF/SWI和SAGA复合物、TFIID和RNA聚合酶II全酶。较小的CCR4-NOT复合物包含CCR4、CAF1、5种NOT蛋白和一些未识别的蛋白。CCR4-NOT蛋白的抑制作用似乎是通过破坏TBP与非规范TATAA序列的关联而发生的，可能是通过它们与TFIIA、TAFs或其他TBP相关因子的直接相互作用。SRB9-11全酶亚复合物也与CCR4-NOT复合物表现出物理和功能上的相互作用。本拨款申请的中心焦点有三个方面：1)两种CCR4-NOT复合物的成分和生理作用是什么？2) CCR4-NOT蛋白如何影响TBP及其相关因子的功能？3)激活转录如何需要CCR4-NOT复合体？1.9 mD CCR4-NOT络合物将被分离，其组分通过质谱分析克隆，并对其功能进行表征。CCR4-NOT复合物影响TBP及其相关因子作用的机制将通过一些生化研究来阐述。将进行遗传筛选，以确定CCR4-NOT复合物与转录重要蛋白之间的新因子或新相互作用。CCR4激活子的功能将通过表征其与SRB9-11、SPT5、TAF60等蛋白独特的C端和n端相互作用来探索。ADH2启动子响应CCR4-NOT复合体的关键特征将被确定，以确定CCR4-NOT复合体如何以积极的方式控制ADH2基因。