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NUCLEOTIDE BINDING PROPERTIES OF SV40 LARGE T PROTEIN

SV40 大 T 蛋白的核苷酸结合特性

基本信息

批准号：
3176091
负责人：
MARGARET K BRADLEY
金额：
$ 17.83万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-07-01 至 1991-06-30
项目状态：
已结题

项目摘要

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地点和SV40Large T的核苷酸结合位点(S)的参数抗原(T-Ag)，并探讨ATPase、 T-Ag的腺苷化等功能。因为有涉及与蛋白质相关的核苷酸的几个活动，我们将首先确定是否存在一个或多个结合位点对于三磷酸腺苷和结合亲和力(S)。我们已经提出了一个中一个一致的ATP结合域的拓扑模型蛋白质，没有结晶学分析的帮助，它包括ATPase和腺化的活性部位。这个研究计划将侧重于测试新提出的在生物化学和遗传上都是模范。至少两个反应性将使用ATP类似物来探测T-Ag中的ATP结合部位，将分析这些修改的特殊性，以及修改后的残留物将被绘制成地图。使用该模型来促进突变的目标，ATP结合位点也将是从基因上进行了探索。将对每个突变的T-AGS进行检测它能够结合和水解三磷酸腺苷，并被腺化。这个这些研究的结果可能支持拟议的模拟或驳斥它们，以及它们的生化特性突变的T-AGS可以揭示两者之间是否存在关联在核苷酸ATPase和腺酰化之间。与相关的 T-Ag突变体，进一步研究生化和/或核苷酸结合活性与蛋白质之间的功能关系蛋白质的其他相关功能将被研究如下作为解旋酶，寡聚，病毒mRNA的自动调节合成、致癌转化和SV40DNA复制。这些调查旨在帮助解决 T-Ag的各个功能相互区别，并有助于了解T-Ag与感染者相互作用的细节- 手机。