GENERAL TRANSCRIPTION FACTORS IN YEAST

这个项目的长期目标是更多地了解真核生物转录启动的基本方面。这一过程在所有真核生物中都是高度保守的，从酵母到人类。这些研究将集中在在酿酒酵母中发现的控制RNA聚合酶II转录启动的蛋白质因子。遗传和生化研究相结合的研究表明，过去研究的几种转录因子，SPT蛋白质，都存在于一个大型蛋白质复合体中。这种复合体被称为SAGA，对于体内正常的RNA聚合酶II的启动至关重要。实验证据有力地表明，SAGA对转录具有不止一种重要的活性。拟议的实验将使用几种方法来分析SAGA在体内的功能。首先，SAGA在启动子中的作用将通过染色质免疫沉淀分析来测试。这些实验将测试关于不同SAGA成分在转录激活中的作用的模型。其次，将在野生型菌株和某些sagA突变体中测量所有mRNAs的体内水平，以确定sagA的全基因组要求。这些实验将使用DNA微阵列技术进行。为了测试SAGA与其他特征转录因子复合体的可能重叠，同样的方法将被用来分析影响一个以上复合体的双突变体。第三，将采用基因方法来识别影响SAGA功能的新突变。其中一个突变筛选将允许测试每个酿酒酵母开放阅读框的缺失。最后，我们将研究与SAGA组件Spt3相关的另一个函数。这个因子，Mot1，不是SAGA的一部分，但有证据表明，Spt3和Mot1都是一般转录因子TATA结合蛋白正常功能所必需的。这些研究应该揭示酵母转录调控的重要方面。鉴于酵母和人类之间的保守性很强，这些研究的结果将适用于人类的转录。这些研究与人类疾病有关，因为人类转录改变与癌症等疾病有很大关系。

The long-term objectives of this project are to learn more about fundamental aspects of transcription initiation in eukaryotes. This process is strongly conserved in all eukaryotes, ranging from yeast to human. The studies will focus on protein factors discovered in the yeast, Saccharomyces cerevisiae, that control transcription initiation by RNA polymerase II. A combination of genetic and biochemical studies have shown that several transcription factors studied in the past, Spt proteins, are all present in a large protein complex. This complex, named SAGA, is critical for normal RNA polymerase II initiation in vivo. Experimental evidence strongly suggests that SAGA has more than one activity important for transcription. The proposed experiments are to use several approaches to analyze SAGA function in vivo. First, the role of SAGA at promoters will be tested by chromatin immunoprecipitation assays. These experiments will test models concerning the roles of different SAGA components in transcriptional activation. Second, the in vivo level of all mRNAs will be measured in wild-type strains and certain SAGA mutants to determine the genome-wide requirements for SAGA. These experiments will be done using DNA microarray technology. To test the possible overlap of SAGA with other characterized transcription factor complexes, the same approach will be used to analyze double mutants that affect more than one complex. Third, genetic approaches will be taken to identify new mutations that affect SAGA function. One of these mutant screens will allow testing deletions of every S. cerevisiae open reading frame. Finally, another function related to one SAGA component, Spt3, will be studied. This factor, Mot1, is not part of SAGA, but evidence suggests that Spt3 and Mot1 are both required for proper function of the general transcription factor, TATA-binding protein. These studies should reveal important aspects of transcriptional control in yeast. Given the strong conservation between yeast and humans, the results from these studies will be applicable to transcription in humans. These studies are relevant to human disease, since altered transcription in humans has been strongly implicated in diseases such as cancer.