FACTORS CONTROLLING PROTEIN SYNTHESIS

控制蛋白质合成的因素

基本信息

批准号：
3269392
负责人：
JOANNE M. RAVEL
金额：
$ 13.82万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
1978
资助国家：
美国
起止时间：
1978-04-01 至 1991-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3269392
关键词：
adenosine triphosphate binding proteins cell free system crosslink density gradient ultracentrifugation eukaryote gel filtration chromatography genetic translation messenger RNA monoclonal antibody nucleic acid structure plant virus protein biosynthesis ribosomes tobacco mosaic virus transfer RNA

项目摘要

The overall objectives of this research are to elucidate the intermediate steps in the initiation of polypeptide synthesis on cytoplasmic ribosomes of eukaryotic cells. The major objectives are to elucidate the mechanism of action of ATP and eukaryotic initiation factors, eIF-3, eIF-4A, eIF-4B, and eIF-4F in the initiation process and to determine what aspects of the primary, secondary or tertiary structure of mRNA affect the efficiency of translation. The specific aims are to: 1) learn more about the physical and functional properties of eIF-3, how the 10 subunits of eIF-3 are arranged, which of the subunits are required for assembly and/or activity, and what the functions of the various subunits are; 2) determine whether there is a physical and/or functional relationship between eIF-4B and eIF-4F; 3) determine whether there are differences in the amounts of eIF-3, 4A, 4B, or 4F required for the initiation of translation of various mRNAs and, if so, how these differences correlate with the primary or postulated secondary or tertiary structures of the mRNAs; 4) determine the role that ATP plays in the interaction of eIF-4A, 4B and 4F with mRNA and in the subsequent binding of mRNA to the small ribosomal subunit; 5) determine the recognition and/or binding site(s) for eIF-4A, 4B and 4F on mRNA. These studies will be carried out with highly purified preparations of eIF-2, 3, 4A, 4B and 4F from wheat germ, and a variety of naturally occurring mRNAs (Alpha and Beta globin, satellite tobacco necrosis virus (STNV), large and small turnip yellow mosaic virus (TYMV), large and small tobacco mosaic virus, cow pea strain (CcTMV)). In addition, truncated or modified forms of these mRNAs will be utilized. Crosslinking reagents and monoclonal antibodies will be used to determine how the subunits of eIF-3 are arranged and what the functions of the subunits are. Polyclonal and monoclonal antibodies to eIF-4B and eIF-4F will be used to determine the structural and functional relationship between these two factors. Deoxyoligonucleotides complementary to sequences in the mRNAs will be used to determine what regions of the mRNA are involved in the recognition and/or binding of the initiation factors and in the hydrolysis of ATP.

本研究的总体目标是阐明中间过程细胞质核糖体上多肽合成的起始步骤真核细胞。主要目标是阐明机制 ATP 和真核起始因子 eIF-3、eIF-4A、eIF-4B 的作用，和 eIF-4F 的启动过程，并确定哪些方面 mRNA 的一级、二级或三级结构影响效率翻译。具体目标是： 1）更多地了解物理知识 eIF-3的功能特性，eIF-3的10个亚基是如何组成的安排了组装和/或活动需要哪些子单元，以及各个亚基的功能是什么； 2）判断是否 eIF-4B 和之间存在物理和/或功能关系 eIF-4F； 3)确定eIF-3的量是否存在差异， 4A、4B 或 4F 是各种 mRNA 翻译起始所需的如果是这样，这些差异如何与主要或假设的相关 mRNA的二级或三级结构； 4）确定角色 ATP 在 eIF-4A、4B 和 4F 与 mRNA 的相互作用中发挥作用，并在随后 mRNA 与核糖体小亚基结合； 5) 确定 mRNA 上 eIF-4A、4B 和 4F 的识别和/或结合位点。这些研究将使用高度纯化的 eIF-2、3、 4A、4B 和 4F 来自小麦胚芽，以及多种天然存在的 mRNA （α 和 β 珠蛋白、卫星烟草坏死病毒 (STNV)、大型和小萝卜黄花叶病毒（TYMV）、大、小烟草花叶病毒病毒，豇豆株（CcTMV））。此外，截断或修改的形式这些 mRNA 的一部分将被利用。交联试剂和单克隆抗体将用于确定 eIF-3 亚基的排列方式以及亚基的功能是什么。多克隆和单克隆 eIF-4B 和 eIF-4F 抗体将用于确定结构以及这两个因素之间的函数关系。将使用与 mRNA 中的序列互补的脱氧寡核苷酸确定 mRNA 的哪些区域参与识别和/或起始因子的结合以及 ATP 的水解。