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

生物化学、分子遗传学和高分辨率X射线结晶学实验将用于 为核糖体和相关因子的作用机制建立结构基础 实现它们在蛋白质合成中的作用和氨基酰基-tRNA合成酶。正确的氨基酸连接到 对的tRNA。为了进一步阐明蛋白质合成过程中每一步的机制，我们希望 为了获得被困在蛋白质合成周期中尽可能多的步骤中的核糖体晶体 并以尽可能高的分辨率建立它们的结构。其结构将是 追求的包括1)结合了m-RNA、tRNA和延伸因子Tu的70年代核糖体的核糖体 2)70年代核糖体与P-位tRNA之间的复合体 含有翻译抑制多肽，以及3)70年代核糖体之间的蛋白质复合体 分泌和膜蛋白插入通道，转位。为了探索两者之间的差异 细菌核糖体及其与真核生物相当大的核糖体，特别是在蛋白质的起始阶段 合成、40S亚基、60S亚基和/或SOS核糖体的结构，包括起始 与IRIS RNA形成的复合体，并使用从酵母或兔网织红细胞中分离出来的核糖体将被 被追捕。为了了解氨酰-tRNA合成酶，如Gln-tRNA合成酶，是如何 刺激tRNA氨酰化反密码子识别，tRNAs络合物的结构 含有突变的反密码子的基因将会被建立。

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.