Characterization of a New Candidate General Transcription Factor

描述（由申请人提供）：酵母转录的研究已经导致许多保守基因的鉴定和表征，这些基因具有转录调节因子的一般作用。被称为Spt选择的遗传选择特别有成效，确定了许多转录调节因子，包括组蛋白、染色质修饰剂、TATA结合蛋白（TBP）、TBP调节因子和转录延伸因子。由于Spt表型指示了在转录过程中发挥作用的蛋白质，我们使用新开发的系统酵母过表达文库来筛选过度表达时导致Spt-表型的基因。我们基本上恢复了之前使用随机基因组文库确定的所有因素，但另外恢复了一个未表征的基因PSH1，该基因编码环结构域蛋白，据报道，在大规模蛋白质组学研究中，该蛋白与FACT组蛋白伴侣复合物和组蛋白共同纯化。我们提出以下具体目标来表征PSH1及其在转录调控中的潜在作用。为了达到特定目的1，我们将确定Psh1的正常功能和过表达表型所需的结构域。Psh1有两个可识别的基序，提示该蛋白的功能作用；一个无名指结构域，以及一段高酸性氨基酸。为了验证这一点，将在关键氨基酸和缺失衍生物中创建点突变，包括Psh1的已识别结构域。这些突变体也将用于识别特定目标2和3中提出的对Psh1的生化活性重要的结构域。对于特定目的2，我们将确定Psh1是否与FACT复合体和组蛋白相互作用，以及它是否与染色质相关。在Spt过表达筛选中发现PSH1表明它在转录中起作用。这个特定目标的目标是获得对其特定功能的生化理解。我们将确定Psh1是否与FACT和组蛋白共ips，染色质共ips将用于评估Psh1与染色质的关联。结合这些方法，将确定Psh1是否与体内FACT、组蛋白和转录区域相关。对于特定目的3，我们将确定Psh1是否泛素化FACT复合体或其他SPT基因。我们最近的初步数据显示重组GST-Psh1在体外具有泛素蛋白连接酶活性。利用在Specific Aim 1中构建的错义和缺失突变体，我们将在体外确定泛素蛋白连接酶活性所需的结构域。在检测了E3泛素蛋白连接酶活性后，下一个目标是鉴定Psh1的体内底物。我们将采取一种候选方法，通过在体内测试底物，如FACT亚基Spt16和Pob3。

DESCRIPTION (provided by applicant): The study of transcription in yeast has led to the identification and characterization of many conserved genes that have general roles as transcriptional regulators. The genetic selection known as the Spt selection has been particularly productive, identifying many transcriptional regulators including histones, chromatin modifiers, TATA binding protein (TBP), regulators of TBP, and transcription elongation factors. Because the Spt phenotype has been indicative of proteins with roles during transcription, we used a newly developed systematic yeast overexpression library to screen for genes that cause the Spt- phenotype when overexpressed. We recovered essentially all of the factors that were previously identified using random genomic libraries, but additionally recovered an uncharacterized gene, PSH1, which encodes a RING domain protein that was reported to co-purify with the FACT histone chaperone complex and histones in large-scale proteomic studies. We propose the following Specific Aims to characterize PSH1 and its potential role in transcription regulation. For specific aim 1 we will determine the domains of Psh1 that are required for its normal function and for its overexpression phenotype. Psh1 has two recognizable motifs that suggest functional roles for this protein; a RING finger domain, and a highly acidic stretch of amino acids. To test this, point mutations in crucial amino acids and deletion derivatives that include the identified domains of Psh1 will be created. These mutants will also serve to identify domains that are important for the biochemical activity of Psh1 as proposed in Specific Aims 2 and 3. For specific aim 2 we will determine if Psh1 interacts with the FACT complex and histones and whether it is associated with chromatin. The identification of PSH1 in the Spt overexpression screen suggests that it has a role in transcription. The goal of this Specific Aim is to gain a biochemical understanding of its specific functions. We will determine whether Psh1 co-IPs with FACT and histones, and chromatin-IPs will be used to evaluate the association of Psh1 with chromatin. Combined, these approaches will establish whether Psh1 is associated with FACT, histones and transcribed regions in vivo. For specific aim 3, we will determine whether Psh1 ubiquitinates the FACT complex or other SPT genes. Our very recent preliminary data shows recombinant GST-Psh1 to possess ubiquitin-protein ligase activity in vitro. Using missense and deletion mutants constructed in Specific Aim 1, we will determine the domains required for ubiquitin-protein ligase activity in vitro. Having detected E3 ubiquitin-protein ligase activity, the next goal is to identify the in vivo substrate(s) of Psh1. We will take a candidate approach by testing in vivo substrates such as the FACT subunits Spt16 and Pob3.