FACTORS CONTROLLING PROTEIN SYNTHESIS

本研究的总体目标是阐明中间体 胞质核糖体启动多肽合成的几个步骤 真核细胞。主要目的是阐明其作用机制。 与真核细胞启动因子eIF-3、eIF-4A、eIF-4B、 和EIF-4F在启动过程中，并确定 信使核糖核酸的一级、二级或三级结构影响基因的作用效率 翻译。具体的目标是：1)了解更多关于体育的知识 以及eIF-3的功能特性，eIF-3的10个亚基是怎样的 排列，组装和/或活动需要哪些亚基， 以及各亚基的功能是什么；2)确定是否 EIF-4B和EIF-4B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， 启动各种mRNAs翻译所需的4A、4B或4F 如果是这样的话，这些差异与主要的或假设的 MRNAs的二级或三级结构；4)决定 ATP在eIF-4A、4B和4F与mRNA的相互作用中发挥作用 随后mRNA与小核糖体亚基的结合；5)决定 EIF-4A、4B和4F的识别和/或结合位点(S)。这些 研究将用高纯度的eIF-2，3， 来自小麦胚芽的4A、4B和4F，以及各种自然产生的mRNAs (阿尔法和贝塔珠蛋白，卫星烟草坏死病毒(STNV)，大和 小芜菁黄花叶病毒(TYMV)、大小烟草花叶病毒 病毒，牛豌豆株(CcTMV))。此外，截断或修改后的表格 其中的mRNAs将被利用。交联剂和单抗 抗体将被用来确定eIF-3亚单位的排列方式 以及亚基的功能是什么。多克隆和单克隆 抗eIF-4B和eIF-4F的抗体将用于确定其结构 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 以确定哪些区域的信使核糖核酸参与识别 和/或启动因子的结合和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.