CONTROL OF EIF-2 ACTIVITY IN REGULATION OF TRANSLATION

翻译的量化控制是翻译过程中的一个重要机制， 基因表达的短期调控。 这是通过改变 mRNA与核糖体结合的速率，并且似乎在水平上受到调节 启动子RNA与核糖体小亚基的相互作用。 真核 起始因子2，eIF-2，促进起始序列的这一步 并且发现在其中所述蛋白质被磷酸化的情况下被可逆地磷酸化。 减少了蛋白质合成的起始。 该机制， eIF-2的磷酸化降低了启动速率，但并不完全 理解，但涉及再循环机制的扰动， 促进因子上的鸟嘌呤核苷酸交换。 虽然 eIF-2磷酸化的重要性已经在 调节网织红细胞和网织红细胞裂解物中的蛋白质合成， 目前尚不清楚这些调节机制在多大程度上 在多种细胞类型中以及在 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， 网织红细胞裂解物中eIF-2的总活性周期。 这些研究 将利用针对eIF-2及其回收蛋白eIF-2B的抗体。 的 另一个需要解决的主要问题是eIF-2在以下方面的作用有多重要： 动物细胞中蛋白质合成的调节。 这些抗体将 加快eIF-2在细胞中代谢的研究， 网织红细胞，并促进评估的程度， eIF-2的修饰用于其它细胞类型。 一系列 培养的兔细胞系将用于记录eIF-2：核糖体和 eIF-2：eIF-2B的比例，相对于细胞类型和增殖率， 稳态条件。 此外，eIF-2与其他 将在改变的情况下确定平移装置的组件。 营养环境 最后，分离的大鼠肝细胞将用于 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.