NUCLEOTIDE BINDING PROPERTIES OF SV40 LARGE T PROTEIN

SV40 大 T 蛋白的核苷酸结合特性

• 批准号: 3176090
• 负责人: MARGARET K BRADLEY
• 金额: $ 17.12万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3176090
• 关键词: DNA binding protein; DNA directed DNA polymerase; adenosine triphosphate; affinity chromatography; binding proteins; covalent bond; gel electrophoresis; genetic mapping; genetic models; high performance liquid chromatography; magnesium; messenger RNA; nucleotides; point mutation; simian virus 40; thin layer chromatography; virus DNA; virus antigen; virus protein; virus replication

The long-term goal of this project is to determine the location and parameters of the nucleotide binding site(s) in SV40 large T antigen (T-Ag), and investigate the relationship between ATPase, adenylylation and other functions of T-Ag. Since there are several activities involving nucleotide associated with the protein, we will first determine whether there is one or more binding sites for ATP and the binding affinity(s). We have proposed a topological model for a consensus ATP binding domain in the protein, without the aid of crystallographic analyses, which includes the active sites for ATPase and adenylation. The research plan will focus on testing aspects of this newly proposed model both biochemically and genetically. At least two reactive ATP analogs will be used to probe the ATP binding site in T-Ag, the modifications will be analyzed for their specificity, and the modified residues will be mapped. Using the model to facilitate the targets for mutagenesis, the ATP binding site will also be probed genetically. Each of the mutant T-Ags will be tested for its ability to bind and hydrolyze ATP, and to be adenylylated. The results of these studies might support aspects of the proposed model or refute them, and the biochemical properties of the mutant T-Ags could reveal whether there is a relationship between nucleotide ATPase and adenylylation. With the relevant T-Ag mutants, further investigation of the biochemical and/or functional relationship between nucleotide binding activities and other associated functions of the protein will be investigated such as helicase, oligomerization, autoregulation of viral mRNA synthesis, oncogenic transformation and SV40 DNA replication. These investigations are intended to help in resolving the individual functions of T-Ag from one another and to help in understanding the details of T-Ag's interactions with the infected- cell.

这个项目的长期目标是确定 SV 40大T细胞中核苷酸结合位点的参数 抗原（T-Ag），探讨ATP酶， 腺苷酸化和T-Ag的其他功能。 既然有 涉及与蛋白质相关的核苷酸的几种活性， 我们将首先确定是否有一个或多个结合位点 对于ATP和结合亲和力。 我们提出了一种 拓扑模型的共识ATP结合结构域在 蛋白质，没有晶体学分析的帮助， 包括ATP酶和腺苷酸化的活性位点。 的 研究计划将集中在测试方面，这一新提出的 生物化学和遗传学的模型。 至少两个反应性 ATP类似物将用于探测T-Ag中的ATP结合位点， 将分析修饰的特异性， 将绘制修饰的残基。 利用模型来促进 诱变的靶点，ATP结合位点也将被 基因检测 每一种突变的T抗原都将被检测 结合和水解ATP以及被腺苷酸化能力。 的 这些研究的结果可能支持拟议的 模型或反驳他们，和生化特性的 突变的T-Ags可以揭示 核苷酸ATP酶和腺苷酸化之间的联系。 有关 T-Ag突变体，进一步研究生化和/或 核苷酸结合活性与 将研究蛋白质的其它相关功能， 如解旋酶、寡聚化、病毒mRNA的自动调节 合成、致癌转化和SV 40 DNA复制。 这些调查旨在帮助解决 T-Ag各自的功能，并帮助 了解T抗原与感染者相互作用的细节 cell.