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

点击翻译按钮获取中文摘要

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.