PROJECT #4: Structural bases of the functions of RNA-protein machines

项目

基本信息

批准号：
7685241
负责人：
THOMAS Arthur STEITZ
金额：
$ 43.82万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7685241
关键词：
Address Amino Acids Amino Acyl Transfer RNA Amino Acyl-tRNA Synthetases Aminoacylation Anti-Bacterial Agents Anticodon Binding Biochemical Biology Catalytic Domain Caulobacter crescentus Complex Development EEF1A1 gene Escherichia coli Eukaryota Glutamine-Specific tRNA Goals Human In Vitro Iris Ligase Membrane Membrane Proteins Molecular Genetics Mutate Nucleotides Oryctolagus cuniculus Peptide Elongation Factor G Peptide Elongation Factor Tu Peptide Initiation Factors Peptides Pharmaceutical Preparations Process Prokaryotic Initiation Factor-2 Protein Biosynthesis Protein Secretion Proteins Resolution Reticulocytes Ribosomes Roentgen Rays Role Side Signal Recognition Particle Site Son of Sevenless Proteins Source Specificity Structure Transfer RNA Transfer RNA Aminoacylation Translating Translation Process Translations Viral Yeasts analog base clinically relevant glutamine-tRNA interest mutant novel peptidyl-tRNA polypeptide protein aminoacid sequence research study

项目摘要

Biochemical, molecular genetic and high resolution X-ray crystallographic experiments will be used to establish the structural bases for the functional mechanisms by which the ribosome and associated factors achieve their roles in protein synthesis and aminoacyl-tRNA synthetases attach .the correct amino acid to the right tRNA. To further illuminate the mechanisms of each step in the process of protein synthesis, we wish to obtain crystals of the ribosome trapped in as many of the steps in the protein synthesis cycle as possible and to establish their structures at the highest resolution possible. The complexes whose structures will be pursued include those of the 1) 70S ribosome with bound m-RNA, tRNA and either elongation factor Tu with aminoacyl-tRNA or elongation factor G, 2) complexes between the 70S ribosomal and a P-site tRNA containing a translation arresting polypeptide, and 3) complexes between the 70S ribosome the protein secretion and membrane protein insertion channel, the translocon. To explore the differences between the bacterial ribosome and its much large counterpart from eukaryotes, particularly in the initiation step of protein synthesis, the structures of the 40S subunit, the 60S subunit and/or the SOS ribosome, including an initiation complex formed with IRIS RNA, and using ribosomes isolated from either yeast or rabbit reticulocyte will be pursued. In order to understand how aminoacyl-tRNA synthetases, such as Gln-tRNA synthetase, are stimulated to aminoacylate the tRNA upon-anticodon recognition, structures of complexes with tRNAs containing mutated anticodons will be established.

生化，分子遗传和高分辨率X射线晶体学实验将用于建立核糖体和相关因素的功能机制的结构基础在蛋白质合成和氨基酰基-TRNA合成酶附着在蛋白质合成中的作用。正确的氨基酸到右tRNA。为了进一步阐明蛋白质合成过程中每个步骤的机制，我们希望为了获得尽可能多的蛋白质合成循环中的步骤中的核糖体晶体并以最高的分辨率建立结构。结构将是追捕包括1）70S核糖体，带有绑定的M-RNA，tRNA和伸长因子TU 氨基酰基tRNA或伸长因子g，2）70S核糖体和P位点tRNA之间的复合物包含捕捉多肽的翻译，3）70S核糖体之间的复合物蛋白质分泌和膜蛋白插入通道，转运。探讨细菌核糖体及其来自真核生物的大量对应物，尤其是在蛋白质的启动步骤中合成，40S亚基的结构，60S亚基和/或SOS核糖体，包括启动用虹膜RNA形成的复合物，使用从酵母或兔网状细胞中分离出的核糖体将是追捕。为了了解氨基酰基-TRNA合成酶（例如GLN-tRNA合成酶）如何刺激以氨基酰基化的tRNA识别tRNA，trnas的复合物的结构将建立含有突变的反登录。