REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITIATION

真核蛋白质合成起始的调控

• 批准号: 2173616
• 负责人: ROBERT E. RHOADS
• 金额: $ 27.57万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-04-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2173616
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; antisense nucleic acid; complementary DNA; genetic regulation; genetic regulatory element; genetic transcription; genetic translation; intermolecular interaction; laboratory rabbit; messenger RNA; molecular cloning; nucleic acid sequence; phosphorylation; posttranslational modifications; protein biosynthesis; protein structure function; recombinant DNA; ribosomes; translation factor

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.

真核生物中的蛋白质合成代表了 遗传信息的表达并说明了很大一部分 蜂窝能量消耗。 对蛋白质合成的理解 它的调节也是对细胞生长的理解至关重要的 作为针对病毒的细胞防御的作用。 长期 该研究项目的目标是了解机制 发生蛋白质合成的哪个开始，集中于速率 - 限制步骤，其中mRNA与核糖体绑定，然后 该过程的调节。 在此期间要追求的具体目标 当前赠款期涉及EIF-4组的启动因子 这催化了这一步骤。 第一个目的涉及结构和 EIF-4E的功能，mRNA帽结合蛋白，其形成的复合物 与其他EIF-4因子，其结合和从48S启动中释放 复杂，以及内部启动的性质。 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。 第三个目的是确定 EIF-4F的结构，这将需要完成主要结构 P220亚基并检查了P220与其他EIF的相互作用 4个多肽。 第四个目标是完成克隆和测序 eIF-4E基因的表征，并表征其上游调节元素。 这些研究将利用传统的生化和分子 生物学方法论以及在 以前的赠款期，包括体外转录/翻译 向量，重组噬菌体，其中包含P220 cDNA和EIF-的一部分 4E基因，哺乳动物细胞表达向量以表达变异蛋白 以及反义RNA，可以阻止蛋白质的启动 在特定阶段的合成和关联常数的测量 通过荧光淬火。