GENERAL TRANSCRIPTION FACTORS IN YEAST

这个项目的长期目标是了解更多关于真核生物转录起始的基本方面。这一过程在所有真核生物中都高度保守，从酵母到人类。 这些研究将集中在酵母中发现的蛋白质因子，酿酒酵母，控制RNA聚合酶II的转录起始。 遗传和生物化学研究的结合表明，过去研究的几种转录因子，Spt蛋白，都存在于一个大的蛋白质复合物中。 这种复合物，命名为佐贺，是体内正常的RNA聚合酶II启动的关键。实验证据有力地表明，佐贺有一个以上的活动，重要的转录。所提出的实验是使用几种方法来分析佐贺在体内的功能。 首先，佐贺在启动子中的作用将通过染色质免疫沉淀试验进行测试。 这些实验将测试关于不同佐贺组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佐贺突变体中测量所有mRNA的体内水平，以确定佐贺的全基因组需求。 这些实验将使用DNA微阵列技术进行。 为了测试佐贺与其他表征的转录因子复合物的可能重叠，将使用相同的方法来分析影响多于一个复合物的双突变体。 第三，将采取遗传方法来鉴定影响佐贺功能的新突变。 这些突变筛选之一将允许测试每个S的缺失。酿酒酵母开放阅读框架。 最后，将研究与佐贺组件Spt3相关的另一个功能。 这个因子Mot1不是佐贺的一部分，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。这些研究应该揭示在酵母转录控制的重要方面。 鉴于酵母和人类之间的强保守性，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，因为人类的转录改变与癌症等疾病密切相关。

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.