REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITIATION

真核生物中的蛋白质合成是蛋白质合成的主要控制点 遗传信息的表达和占相当大的比例 细胞能量消耗的比例。对蛋白质合成的认识 而它的调控也是理解细胞生长的核心 作为对病毒的行动和细胞防御。长期的 本研究项目的目的是通过以下方式了解该机制 蛋白质合成的启动，关注的是速度- 限制步骤，其中mRNA与核糖体结合，并且 对这一过程的监管。在此期间要实现的具体目标 目前的授权期涉及eIF-4组的启动因素 它催化了这一步。第一个目标涉及结构和 MRNA帽结合蛋白eIF-4E的功能及其形成的复合体 与其他eIF-4因子一起，其结合和释放从48S开始 复杂性，以及内在启蒙的本质。第二个目标是 了解eIF-4E在Ser-53上磷酸化的机制 刺激蛋白质合成。第三个目标是确定 EIF-4F的结构，这将需要完成初级结构 并检测p220与其他eIF的相互作用。 4多肽。第四个目标是完成克隆和测序 目的是研究eIF-4E基因及其上游调控元件的特性。 这些研究将利用传统的生物化学和分子生物学 生物学方法论以及在 前一批款期，包括体外转录/翻译 载体，重组噬菌体含有p220基因和eIF的部分片段。 4E基因、哺乳动物细胞表达载体表达差异蛋白 除了反义RNA，还有阻止蛋白质启动的能力 特定阶段的合成及缔合常数的测定 通过荧光猝灭。

Protein synthesis in eukaryotes represents a major control point for the expression of genetic information and accounts for a substantial fraction of cellular energy expenditure. An understanding of protein synthesis and its regulation is central to an understanding of cell growth as well as the action of and cellular defenses against viruses. The long-term objectives of this research project are to understand the mechanism by which initiation of protein synthesis occurs, focussing on the rate- limiting step wherein mRNA becomes bound to the ribosome, and the regulation of this process. The specific aims to be pursued during the current grant period involve the initiation factors of the eIF-4 group which catalyze this step. The first aim deals with the structure and function of eIF-4E, the mRNA cap-binding protein, the complexes it forms with other eIF-4 factors, its binding and release from the 48S initiation complex, and the nature of internal initiation. The second aim is to understand the mechanism by which phosphorylation of eIF-4E at Ser-53 stimulates protein synthesis. The third aim is to determine the structure of eIF-4F, which will entail completing the primary structure of the p220 subunit and examining the interaction of p220 with other eIF- 4 polypeptides. The fourth aim is to complete the cloning and sequencing of the eIF-4E gene and to characterize its upstream regulatory elements. These studies will make use of traditional biochemical and molecular biological methodology as well as a variety of tools developed during the previous grant period, including in vitro transcription/translation vectors, recombinant phage containing portions of the p220 cDNA and eIF- 4E gene, mammalian cell expression vectors to express variant proteins as well as antisense RNA, the ability to arrest initiation of protein synthesis at specific stages and the measurement of association constants by fluorescence quenching.