Eukaryotic Gene Regulation as Studied in Yeast

描述（由申请人提供）：基因表达的调控是不同生物过程的基础，包括从受精卵到生物体的发育，生物体对细胞外信号的反应，以及记忆的根深蒂固。这笔拨款要求继续我们对基因调控机制的分析，这将有助于我们理解正常的过程，并阐明它们是如何出错的。拟议的研究主要使用酵母细胞，一种真核生物，其中基因调控的许多方面与哺乳动物细胞中的基因调控密切相关。在一系列论文和书籍中，我们开发了一个模型——称为“招募”模型——用于转录调节因子如何决定任何给定基因是被激活还是被抑制。我们在这里提出了一系列实验，以各种方式探索和测试这个模型。我们已经开发了一种方法来测量各种蛋白质复合物的外观，当它们被dna结合激活剂招募时，在特定基因处。超过100种蛋白质必须被带到基因中才能进行转录，我们正在用我们的方法研究这个大的复合体是如何逐步形成的。在相关的发展中，我们现在可以非常准确地测定核小体在DNA上的配置，我们正在研究核小体的定位和移除如何受到其他DNA结合蛋白的影响。由于尚不清楚的原因，在高等真核生物中，当转录激活子与基因的数百个碱基对结合时，它可以开启基因的转录，但在酵母中，激活子必须与基因结合得更近才能有效地工作。我们已经对酵母进行了修饰，使其能够“远距离激活”，现在我们正在分析其机制，看看效果是否与招募模型一致。我们正在研究一种蛋白质的特性，这种蛋白质似乎具有一种独特的能力，当人工招募到一个基因中时，它可以触发基因表达。特别是，我们正在测试这样一种想法，即这种蛋白质，一种称为中介的大型复合体的组成部分，在细胞中迅速降解，这种不稳定性促进了转录的激活

DESCRIPTION (provided by applicant): Regulation of gene expression underlies disparate biological processes, including development of an organism from a fertilized egg, the response of organisms to extra-cellular signals, and the ingraining of memories. The grant requests funds to continue our analyses of mechanisms of gene regulation that will help us understand the normal processes and shed light on how they can go awry. The proposed studies use primarily yeast cells, a eukaryote in which many aspects of gene regulation are closely related to gene regulation in mammalian cells. In a series of papers and books we have developed a model - called the 'recruitment' model - for how transcriptional regulators determine whether any given gene will be activated or repressed. We propose here a series of experiments that probe and test this model in various ways. We have developed methods to measure the appearance, at a specific gene, of various protein complexes as they are recruited by a DNA-bound activator. Over 100 proteins must be brought to the gene for transcription to proceed, and we are using our methods to study how this large complex is formed stepwise. In a related development, we now can assay with great accuracy the disposition of nucleosomes on DNA, and we are studying how nucleosome positioning and removal is effected by other DNA binding proteins. For reasons that are not understood, a transcriptional activator can turn on transcription of a gene when bound many hundreds of base pairs from the gene in higher eukaryotes, but in yeast the activator must be bound much closer to the gene to work efficiently. We have modified yeast so as to allow 'activation at a distance', and we are now analyzing the mechanism to see whether the effect is consistent with the recruitment model. And we are examining the properties of a protein that seems to have a unique ability to trigger gene expression when artificially recruited to a gene. In particular we are testing the idea that this protein, a component of the large complex called the Mediator, is rapidly degraded in cells, and that this instability facilitates activation of transcription