CONTROL OF EIF-2 ACTIVITY IN REGULATION OF TRANSLATION

翻译的数量控制是翻译过程中的重要机制 基因表达的短期调节。它是通过改变 MRNA与核糖体结合的速率，似乎是在 启动子RNA与核糖体小亚基的相互作用。真核生物 启动因子2，eIF-2，促进启动序列的这一步 并被发现在以下情况下被可逆地磷酸化 蛋白质合成的启动减少了。这是一种机械主义 EIF-2的磷酸化不能完全降低起始率 理解，但涉及回收机制的扰动， 促进因子上的鸟嘌呤核苷酸交换。尽管 EIF-2磷酸化的重要性已经在 网织红细胞和网织红细胞裂解物中蛋白质合成的调节， 目前尚不清楚这些监管机制在多大程度上代表着 可在各种细胞类型中发现的一般机制 不同的生理条件。 本提案承诺完成对 磷酸化降低eIF-2活性的机制及其研究进展 网织红细胞裂解液中eIF-2的总活性周期。这些研究 将利用针对eIF-2及其循环蛋白eIF-2B的抗体。这个 另一个需要解决的主要问题是eIF-2在 动物细胞中蛋白质合成的调节。抗体会 加快研究eIF-2在其他细胞中的代谢 网织红细胞和促进评估的程度 EIF-2的修饰也被用于其他类型的细胞。一系列 培养的兔细胞株将用于记录eIF-2：核糖体和 EIF-2：eIF-2B与细胞类型和增殖率的比率 稳定状态条件。此外，eIF-2与其他 平移装置的部件将在更改时确定 营养环境。最后，分离的大鼠肝细胞将用于 通过改变评估eIF-2参与蛋白质合成的调节 代谢物水平和激素信号。

The quantitative control of translation is an important mechanism in the short-term regulation of gene expression. It is achieved by changes in the rate of mRNA binding to ribosomes and seems to be regulated at the level of initiator RNA interaction with the small ribosomal subunit. Eukaryotic initiation factor 2, eIF-2, promotes this step of the initiation sequence and is found to be reversibly phosphorylated in circumstances in which the initiation of protein synthesis is reduced. The mechnism which phosphorylation of eIF-2 reduces the rate of initiation is not completely understood, but involves the perturbation of the recycling mechanism which promotes guanine nucleotide exchange on the factor. Although the importance of eIF-2 phosphorylation has been clearly demonstrated in the regulation of protein synthesis in reticulocytes and reticulocyte lysate, it is not known to what extent these regulatory mechanisms represent general mechanisms that may be found in a variety of cell types and under varying physiological conditions. The present proposal undertakes to complete the investigations on the mechanisms by which phosphorylation reduces eIF-2 activity and to work out the overall activity cycle of eIF-2 in reticulocyte lysate. These studies will utilize antibodies to eIF-2 and its recycling protein, eIF-2B. The other major question to be addressed is how important a role eIF-2 plays in the regulation of protein synthesis in animal cells. The antibodies will expedite the investigation of eIF-2 metabolism in cells other than reticulocytes and facilitate the assessment of the extent to which modification of eIF-2 is employed in other cell types. A series of cultured rabbit cell lines will be used to document eIF-2:ribosome and eIF-2:eIF-2B ratios with respect to cell type and proliferation rate under steady state conditions. In addition, the association of eIF-2 with other components of the translational apparatus will be determined under changing nutrient environments. Finally isolated rat hepatocytes will be used to assess eIF-2 involvement in the regulation of protein synthesis by changing metabolite levels and hormonal signals.