YEAST GENES INVOLVED IN GENERAL TRANSCRIPTIONAL CONTROL

本研究的目的是阐明真核基因调控的机制。选择研究的系统是多蛋白CCR4-NOT复合体，它对基因表达既有积极的影响，也有消极的影响。它由两个复合体组成，大小分别为1.9和1.0md，不同于其他转录上重要的蛋白质组，如SNF/SWI和SAGA复合体、TFIID和RNA聚合酶II全酶。较小的CCR4-NOT复合体包含CCR4、CAF1、五个NOT蛋白和几个未知蛋白。CCR4-NOT蛋白的抑制作用似乎是通过破坏TBP与非规范TATAA序列的结合而发生的，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而发生的。SRB9-11全酶亚复合体也与CCR4-NOT复合体显示出物理和功能上的相互作用。这项资助提案的中心焦点有三个：1)两个CCR4-NOT复合体的组成和生理作用是什么？2)CCR4-NOT蛋白如何影响TBP及其相关因子的功能？3)CCR4-非复合体是如何激活转录所必需的？将分离出1.9md的CCR4-NOT复合体，通过质谱分析克隆其组分，并对其功能进行鉴定。CCR4-NOT复合体影响TBP的作用及其相关因素的机制将通过几项生化研究加以阐述。将进行遗传筛选，以确定CCR4之间的新因素或新的相互作用--不是复杂的和转录上重要的蛋白质。CCR4激活剂的功能将通过鉴定其与SRB9-11、SPT5、TAF60和其他蛋白质的独特的C-端和N-端相互作用来探索。将确定对CCR4-NOT复合体反应的ADH2启动子的关键特征，以确定CCR4-NOT复合体如何以积极的方式控制ADH2基因。

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.