EXTRAGENIC SUPPRESSORS OF THE TATA-BINDING PROTEIN

TATA 结合蛋白的外源抑制子

基本信息

批准号：
2634696
负责人：
Martin C Schmidt
金额：
$ 22.59万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-07-01 至 2000-05-31
项目状态：
已结题

项目摘要

The long-term objectives of the study are to define in molecular detail the mechanisms by which proteins regulate gene expression through direct interaction with the TATA-binding protein (TBP). TBP plays a central role in all eukaryotic gene expression since it is required for transcription initiation by all three nuclear RNA polymerase enzymes. A number of human and viral proteins that bind TBP, including the p53, myc and E1A proteins, have been implicated in transcriptional control and tumorigenesis. Thus an understanding of the means by which proteins bind TBP and regulate gene expression will have broad implications on diverse biological processes including growth control and cancer. We propose to investigate the mechanism by which the Std1 protein regulates gene expression as a paradigm for other transcriptional regulators that bind TBP. The STD1 gene was isolated as an extragenic suppressor of a dominant negative mutation in TBP. Std1p directly binds TBP as determined in both genetic and biochemical assays. Std1p enhances the DNA binding activity of TBP in a promoter specific fashion. Work from our lab and others indicate that Std1p activates gene expression but is not a typical transcriptional activator. Std1p does not contain a functional activation domain. Its binding site is not upstream of the TATA element, but overlaps the TATA element. All of these data combined suggest that the Std1 protein may regulate transcription by a mechanism that is fundamentally different from other transcriptional activators. Five specific aims are proposed. l) Characterize the Std1p-mediated regulation of TBP. The effect of Std1p on the kinetics, temperature dependence and site specificity of TBP-DNA and an intermediate of preinitiation complex formation (DAB) will be determined. The sequence specificity of Std1p's DNA binding activity and the role it plays in the regulation of the DNA binding activity of TBP will be determined. 2) The effect of Std1p on transcription initiation will be determined by nuclease S1 analysis of mRNA's from the SUC2 gene in vivo and from transcription reactions in vitro will be used to determine whether the Std1p alters the start site selection or increases the frequency of transcription initiation at the SUC2 promoter. 3) Identify and characterize the domains of Std1p that specify its known biochemical activities. These studies will use site specific mutagenesis of the protein and analysis of its biochemical properties in vitro and relate these activities with its function in vivo. 4) Isolate extragenic suppressors of std1 null alleles as a means to identify other protein that function in the same biochemical path way in vivo. 5) Isolate and characterize additional TBP-binding proteins as extragenic suppressors of the dominant negative allele of TBP used to isolate STD1. Also, temperature sensitive alleles of TBP with lesions in helix H2' will be screened for extragenic suppressors.

该研究的长期目标是定义分子细节蛋白质通过直接调节基因表达的机制与TATA结合蛋白（TBP）的相互作用。 TBP扮演着核心角色在所有真核基因的表达中，由于需要转录所有三种核RNA聚合酶的启动。许多人和结合TBP的病毒蛋白，包括p53，myc和e1a蛋白，已与转录控制和肿瘤发生有关。因此了解蛋白质结合TBP并调节基因的手段表达将对多种生物学过程具有广泛的影响包括生长控制和癌症。我们建议调查 STD1蛋白调节基因表达为A的机制用于结合TBP的其他转录调节器的范例。 STD1基因被分离为显性阴性突变的基本抑制剂在TBP中。 STD1P直接结合遗传和遗传和生化测定。 STD1P增强了TBP的DNA结合活性发起人特定的时尚。来自我们实验室的工作，其他工作表明 STD1P激活基因表达，但不是典型的转录激活剂。 STD1P不包含功能激活域。它是绑定位点不是塔塔元素的上游，而是塔塔（Tata）重叠元素。所有这些数据的总和表明STD1蛋白可能通过与从根本上不同的机制调节转录其他转录激活剂。提出了五个具体目标。 l）表征STD1P介导的 TBP的调节。 STD1P对动力学，温度的影响 TBP-DNA的依赖性和位点特异性和一个中间体将确定前启动复合物的形成（DAB）。序列 STD1P的DNA结合活性及其在将确定TBP的DNA结合活性的调节。 2） STD1P对转录起始的影响将由核酸酶确定从体内和转录的SUC2基因的S1分析mRNA分析体外反应将用于确定STD1P是否改变启动现场选择或增加转录频率在SUC2启动子处启动。 3）识别和表征域 STD1P指定其已知的生化活动。这些研究会使用位点特异性诱变的蛋白质及其分析体外的生化特性，并将这些活动与之联系在体内功能。 4）STD1 NULL等位基因的隔离基因抑制剂作为识别在同一生化中起作用的其他蛋白质的手段体内路径路。 5）隔离和表征其他TBP结合蛋白质作为TBP的主要负等位基因的外部抑制剂用于隔离STD1。另外，TBP的温度敏感等位基因螺旋H2'中的病变将被筛选为外部抑制器。