FACTORS CONTROLLING PROTEIN SYNTHESIS

本研究的总体目标是阐明中间体 细胞质核糖体上多肽合成起始的步骤 真核细胞。 主要目的是阐明机制 ATP和真核起始因子eIF-3，eIF-4A，eIF-4 B， 和eIF-4F在启动过程中，并确定哪些方面的 mRNA的一级、二级或三级结构会影响 翻译. 具体目标是：1）了解更多关于物理 以及eIF-3的功能特性，eIF-3的10个亚基是如何 布置，组装和/或活动需要哪些子单元， 以及各个子单元的功能; 2）确定是否 在eIF-4 B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、4 B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 mRNA的二级或三级结构; 4）确定 ATP在eIF-4A、4 B和4F与mRNA的相互作用中起作用， 随后mRNA与核糖体小亚基的结合; 5）确定 mRNA上eIF-4A、4 B和4F的识别和/或结合位点。 这些 将用eIF-2，3， 4A、4 B和4F，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， 小萝卜黄花叶病毒（TYMV）、烟草大、小花叶病毒 病毒，豇豆株（CcTMV））。 此外，截断或修改的形式 将使用这些mRNA。 交联剂和单克隆抗体 抗体将用于确定eIF-3亚基的排列方式 以及子单元的功能是什么。 多克隆和单克隆 eIF-4 B和eIF-4F的抗体将用于确定结构 以及两者之间的函数关系。 将使用与mRNA序列互补的脱氧寡核苷酸 以确定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。

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.