EXTRAGENIC SUPPRESSORS OF THE TATA-BINDING PROTEIN

这项研究的长期目标是从分子上详细定义 蛋白质通过直接调控基因表达的机制 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， 所有三种核RNA聚合酶的启动。许多人类 和结合TBP的病毒蛋白，包括p53、myc和E1 A蛋白， 与转录控制和肿瘤发生有关。因而 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 Std 1蛋白作为一种调节基因表达的机制， 其他转录调节因子结合TBP的范例。STD 1基因 作为显性负突变的基因外抑制基因被分离出来 在TBP。Std 1 p直接结合TBP，如在遗传和 生化分析Std 1 p增强TBP的DNA结合活性， 启动子特异性方式。我们实验室和其他机构的研究表明， Std 1 p激活基因表达，但不是典型的转录 活化剂。Std 1 p不包含功能性激活结构域。其 结合位点不在TATA元件的上游，但与TATA元件重叠 元素所有这些数据结合起来表明，Std 1蛋白可能 调节转录的机制是根本不同的， 其他转录激活因子。 提出了五个具体目标。l）表征Std 1 p介导的 TBP规则。Std 1 p对动力学、温度的影响 TBP-DNA的依赖性和位点特异性以及 将测定预引发复合物形成（DAB）。序列 Std 1 p的DNA结合活性的特异性及其在 将确定TBP的DNA结合活性的调节。2)的 Std 1 p对转录起始的影响将通过核酸酶来确定 来自体内SUC 2基因和来自转录的mRNA的S1分析 体外反应将用于确定Std 1 p是否改变了 启动位点选择或增加转录频率 在SUC 2启动子处起始。3)识别和表征域 的Std 1 p，说明其已知的生化活性。这些研究将 使用蛋白质的位点特异性突变并分析其 体外生物化学性质，并将这些活动与其 在体内发挥作用。4)分离std 1无效等位基因的基因外抑制因子 作为一种手段，以确定其他蛋白质的功能，在同一生化 体内途径。 5)分离并表征额外的TBP结合 TBP显性负等位基因的基因外抑制蛋白 用于分离STD 1。此外，TBP的温度敏感等位基因， 螺旋H2'中的损伤将被筛选以寻找基因外抑制因子。

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.