Eukaryotic Gene Regulation as Studied in Yeast

描述(申请人提供)：基因表达调控是不同的生物学过程的基础，包括从受精卵发育成生物体，生物体对细胞外信号的反应，以及记忆的根深蒂固。这笔拨款要求资金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，并阐明它们如何会出错。拟议的研究主要使用酵母细胞，这是一种真核生物，其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。在一系列的论文和书籍中，我们开发了一个模型--称为‘招募’模型--关于转录调节器如何决定某个特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，以不同的方式探索和测试这一模型。我们已经开发出方法来测量不同蛋白质复合体在特定基因上的外观，因为它们被DNA结合的激活剂所招募。超过100种蛋白质必须被带到基因上才能进行转录，我们正在使用我们的方法来研究这个大型复合体是如何逐步形成的。在一个相关的发展中，我们现在可以非常准确地分析核小体在DNA上的分布，我们正在研究其他DNA结合蛋白如何影响核小体的定位和移除。由于未知的原因，在高等真核生物中，当转录激活剂与基因的数百个碱基对结合时，可以启动基因的转录，但在酵母中，激活剂必须与基因结合得更近才能有效地发挥作用。我们已经对酵母菌进行了改造，使其能够在一定距离内被激活，目前我们正在分析其作用机制，以确定其效果是否与招募模型一致。我们正在研究一种蛋白质的性质，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上时，它就会触发基因表达。特别是，我们正在测试这样一种想法，即这种蛋白质是被称为介体的大型复合体的一种成分，在细胞中迅速降解，这种不稳定有助于转录的激活

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