Characterization of a New Candidate General Transcription Factor

新候选通用转录因子的表征

• 批准号: 7741728
• 负责人: Grace Marie Jones
• 金额: $ 3.86万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-18 至 2010-06-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7741728
• 关键词: Amino Acids; Biochemical; Biological Assay; Chromatin; Classification; Complex; Data; Elongation Factor; Fingers; General Transcription Factors; Genes; Genetic; Genetic Transcription; Genomic Library; Goals; Histones; Libraries; Molecular Chaperones; Phenotype; Point Mutation; Proteins; Proteomics; Recombinants; Reporting; Role; Stretching; TATA-Box Binding Protein; Tertiary Protein Structure; Testing; Transcription Elongation; Transcriptional Regulation; Ubiquitin-Protein Ligase Complexes; Yeasts; genetic selection; in vitro activity; in vivo; mutant; overexpression; ubiquitin-protein ligase

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.

描述（由申请人提供）：对酵母中转录的研究已经导致了许多保守基因的鉴定和表征，这些基因具有作为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，鉴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的调节因子和转录延伸因子。因为Spt表型已经指示在转录过程中具有作用的蛋白质，所以我们使用新开发的系统性酵母过表达文库来筛选当过表达时引起Spt表型的基因。我们基本上回收了所有以前使用随机基因组文库鉴定的因子，但另外回收了一个未表征的基因PSH 1，该基因编码一种RING结构域蛋白，据报道，该蛋白在大规模蛋白质组学研究中与FACT组蛋白伴侣复合物和组蛋白共纯化。我们提出以下具体目标来表征PSH 1及其在转录调控中的潜在作用。对于具体目标1，我们将确定其正常功能和过表达表型所需的Psh 1的结构域。Psh 1有两个可识别的基序，表明这种蛋白质的功能作用;一个环指结构域和一段高度酸性的氨基酸。为了测试这一点，将创建关键氨基酸中的点突变和缺失衍生物，包括Psh 1的鉴定结构域。这些突变体还将用于鉴定对特定目的2和3中提出的Psh 1的生化活性重要的结构域。对于具体目标2，我们将确定Psh 1是否与FACT复合物和组蛋白相互作用，以及它是否与染色质相关。在Spt过表达筛选中鉴定PSH 1表明其在转录中起作用。本课程的目标是从生物化学的角度了解它的具体功能。我们将确定Psh 1 co-IP与FACT和组蛋白以及染色质-IP是否用于评价Psh 1与染色质的相关性。结合这些方法将确定Psh 1是否与体内FACT、组蛋白和转录区域相关。对于具体目标3，我们将确定Psh 1是否泛素化FACT复合物或其他SPT基因。我们最近的初步数据显示，重组GST-Psh 1在体外具有泛素蛋白连接酶活性。使用错义和缺失突变体构建的具体目标1，我们将确定所需的结构域的泛素蛋白连接酶活性在体外。在检测到E3泛素蛋白连接酶活性后，下一个目标是鉴定Psh 1的体内底物。我们将通过测试体内底物（如FACT亚基Spt 16和Pob 3）采取候选方法。