Eukaryotic Gene Regulation as Studied in Yeast

描述（由申请人提供）：基因表达的调节是不同生物过程的基础，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内化。该基金要求资金继续我们的基因调控机制的分析，这将有助于我们了解正常的过程，并阐明他们是如何出错的。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。在一系列的论文和书籍中，我们开发了一个称为“招募”模型的模型，用于研究转录调节因子如何决定任何给定的基因是被激活还是被抑制。我们在这里提出了一系列的实验，以各种方式探测和测试这个模型。我们已经开发了方法来测量的外观，在一个特定的基因，各种蛋白质复合物，因为它们被招募的DNA结合的激活剂。超过100种蛋白质必须被带到基因中进行转录，我们正在使用我们的方法来研究这个大的复合物是如何逐步形成的。在一个相关的发展中，我们现在可以非常准确地测定核小体在DNA上的分布，我们正在研究核小体的定位和去除如何受到其他DNA结合蛋白的影响。由于尚不清楚的原因，转录激活因子在高等真核生物中与基因结合数百个碱基对时可以启动基因的转录，但在酵母中，激活因子必须与基因更紧密地结合才能有效地工作。我们已经修改了酵母，以便允许“远距离激活”，我们现在正在分析机制，看看效果是否与招聘模型一致。我们正在研究一种蛋白质的特性，这种蛋白质似乎有一种独特的能力，当人工招募到一个基因时，它会触发基因表达。特别是，我们正在测试这样一种想法，即这种蛋白质是称为介体的大复合体的一个组成部分，在细胞中迅速降解，这种不稳定性有助于转录的激活

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