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

项目

• 批准号: 8052873
• 负责人: THOMAS Arthur STEITZ
• 金额: $ 44.34万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8052873
• 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

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.

生物化学、分子遗传学和高分辨率X射线晶体学实验将用于 为核糖体和相关因子的功能机制建立结构基础 完成它们在蛋白质合成中的作用，氨酰-tRNA合成酶将正确的氨基酸连接到蛋白质上。 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 为了获得在蛋白质合成周期中尽可能多的步骤中捕获的核糖体晶体， 并以尽可能高的分辨率建立它们的结构。其结构将被 所追求的包括那些1）70 S核糖体与结合的m-RNA，tRNA和延伸因子Tu， 氨酰-tRNA或延伸因子G，2）70 S核糖体和P-位点tRNA之间的复合物 含有翻译阻滞多肽，和3）70 S核糖体与蛋白质之间的复合物 分泌和膜蛋白插入通道，即易位子。为了探索 细菌核糖体和真核生物的大得多的对应物，特别是在蛋白质的起始步骤中， 合成，40 S亚基、60 S亚基和/或SOS核糖体的结构，包括起始 与IRIS RNA形成的复合物，并使用从酵母或兔网织红细胞中分离的核糖体， 追求。为了了解氨酰-tRNA合成酶，如Gln-tRNA合成酶， 在反密码子识别的情况下，刺激氨酰化tRNA，与tRNA的复合物的结构 含有突变的反密码子。