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，并占很大一部分 细胞能量消耗。 对蛋白质合成的理解 它的调节也是理解细胞生长的核心 细胞对病毒的防御。 长期 本研究项目的目标是通过以下方式了解该机制： 蛋白质合成的起始阶段，关注的是 限制步骤，其中mRNA与核糖体结合，并且 规范这个过程。 会议期间要实现的具体目标 目前的资助期涉及eIF-4集团的启动因素 催化这一步。 第一个目标涉及结构和 mRNA帽结合蛋白eIF-4 E的功能及其形成的复合物 与其他eIF-4因子，其结合和释放从48 S起始 复杂性，以及内部启动的性质。 第二个目标是 了解eIF-4 E的Ser-53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，这将需要完成主要结构 p220亚基的表达，并检测p220与其他eIF- 4种多肽。 第四个目标是完成克隆和测序 的eIF-4 E基因，并表征其上游调控元件。 这些研究将利用传统的生物化学和分子生物学方法， 生物学方法以及各种工具， 上一个赠款期，包括体外转录/翻译 载体，含有p220 cDNA和eIF-的部分的重组噬菌体， 4 E基因，表达变体蛋白的哺乳动物细胞表达载体 以及反义RNA，阻止蛋白质起始的能力 特定阶段的合成和缔合常数的测定 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.