EXTRAGENIC SUPPRESSORS OF THE TATA-BINDING PROTEIN

TATA 结合蛋白的外源抑制子

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/2634696
• 关键词: DNA binding protein; alleles; chimeric proteins; chromatin; conformation; fungal genetics; gene dosage; genetic promoter element; genetic regulation; genetic transcription; human genetic material tag; micromanipulator; molecular genetics; nucleic acid sequence; nucleic acid structure; oligonucleotides; polymerase chain reaction; regulatory gene; site directed mutagenesis; transcription factor

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详细定义分子细节 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发挥核心作用 在所有真核基因表达中，因为它是转录所必需的 由所有三种核RNA聚合酶引发。若干人类 以及结合 TBP 的病毒蛋白，包括 p53、myc 和 E1A 蛋白， 与转录控制和肿瘤发生有关。因此 了解蛋白质结合 TBP 和调节基因的方式 表达将对不同的生物过程产生广泛的影响 包括生长控制和癌症。我们建议调查 Std1 蛋白调节基因表达的机制 其他结合 TBP 的转录调节因子的范例。 STD1基因 被分离为显性失活突变的基因外抑制子 待定。 Std1p 直接结合 TBP，这在遗传和 生化测定。 Std1p 增强 TBP 的 DNA 结合活性 发起人特定的时尚。我们实验室和其他实验室的工作表明 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 结合位点不在 TATA 元件上游，但与 TATA 重叠 元素。所有这些数据综合表明 Std1 蛋白可能 通过一种根本不同的机制来调节转录 其他转录激活剂。 提出了五个具体目标。 l) 表征 Std1p 介导的 TBP 的监管。 Std1p对动力学、温度的影响 TBP-DNA 和中间体的依赖性和位点特异性 将确定预引发复合物形成（DAB）。顺序 Std1p DNA 结合活性的特异性及其在 TBP的DNA结合活性的调节将被确定。 2) 的 Std1p 对转录起始的影响将由核酸酶决定 对体内 SUC2 基因和转录的 mRNA 进行 S1 分析 体外反应将用于确定 Std1p 是否改变 开始位点选择或增加转录频率 在 SUC2 启动子处启动。 3) 识别并表征域 Std1p 指定其已知的生化活性。这些研究将 使用蛋白质的位点特异性诱变及其分析 体外生化特性并将这些活性与其相关 在体内发挥作用。 4) 分离 std1 无效等位基因的外源抑制子 作为识别在相同生化中发挥作用的其他蛋白质的手段 体内途径。 5) 分离并表征额外的 TBP 结合 作为 TBP 显性失活等位基因的外源抑制子的蛋白质 用于隔离 STD1。此外，TBP 的温度敏感等位基因 将筛选螺旋 H2' 中的损伤是否存在外源抑制因子。