Program in Macromolecular Structure, Motion, Control

高分子结构、运动、控制程序

• 批准号: 7297746
• 负责人: THOMAS Arthur STEITZ
• 金额: $ 22.45万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-25 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7297746
• 关键词: Halobacteriaceae; X ray crystallography; chemical structure; conformation; genetic recombination; hydrolysis; intermolecular interaction; macromolecule; messenger RNA; molecular assembly /self assembly; molecular dynamics; nucleic acid sequence; ribosomes; transfer RNA

Biochemical, molecular genetic and high resolution X-ray crystallographic experiments will be used to establish the structural bases for the functional mechanisms of proteins (and enzymes) that interact with DNA or RNA, with a major emphasis on large macromolecular assemblies. Of particular interest are the proteins and nucleic acids involved in facilitating the processes of replication, gene'expression and recombination - the Central Dogma of Molecular Biology. 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) the complex of the H. marismortui SOS subunit with fragments of the signal recognition particle, and 3) complexes between either the 70S ribosome or its SOS subunit complexed with 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, the structures of the 40S subunit, the 60S subunit and/or the 80S ribosome, including an initiation complex formed with IRIS RNA, and using ribosomes isolated from either yeast or rabbit reticulocyte will be pursued. To explore the structural basis of antibiotic specificity, the RNA forming the peptidyl transferase center of H. marismortui 50 S subunit will be replaced by RNA sequences corresponding to those of eubacterial or eukaryotic large subunit rRNA and their complexes with, antibiotics studied. To further understand the mechanism by which the site specific recombinase, gamma-delta resolvase, forms a synaptic complex with two 115 base-pair DNAs and achieves recombination, the structure of the entire 12 subunit complex with DNA will be determined using mutant gamma-delta resolvase. To gain insights into m-RNA splicing the structure of group II intron self-splicing RNA will be pursued. In order to explore the alternate pathway of producing cys-tRNACys used by some archaea, the structure of the archaeal ortholog of Phe-tRNA synthetase that amino acylates tRNACys with phosphoserine will be established with the appropriate substrates bound.

生化，分子遗传和高分辨率X射线晶体学实验将用于 建立与DNA或DNA相互作用的蛋白质（和酶）功能机理的结构碱基 RNA，主要重点是大分子分子组件。特别感兴趣的是蛋白质和 参与促进复制过程，基因表达和重组过程的核酸 - 中央 分子生物学的教条。进一步阐明蛋白质过程中每个步骤的机制 合成，我们希望获得蛋白质合成循环中许多步骤中被困在许多步骤中的核糖体的晶体 尽可能地以最高分辨率建立其结构。结构将会 被追捕包括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）H。marismortui SOS亚基的复合物与碎片的碎片 信号识别粒子和3）70S核糖体或其SOS亚基之间的复合物 蛋白质分泌和膜蛋白插入通道，转运。探索差异 细菌核糖体及其来自真核生物的大量对应物，40年代亚基的结构，60年代 亚基和/或80年代核糖体，包括用虹膜RNA形成的启动复合物，并使用核糖体 从酵母或兔子网状细胞中分离出来。探索抗生素的结构基础 特异性，形成H. marismortui 50 S亚基的肽基转移酶中心的RNA将被取代 与细菌或真核大型亚基rRNA及其复合物相对应的RNA序列 抗生素研究了。为了进一步了解位点特异性重组酶，伽马 - 戴尔塔分辨率的机制， 形成一个具有两个115碱基对DNA的突触复合物并实现重组，整个12的结构 与DNA的亚基复合物将使用突变伽马 - 戴尔塔分辨率确定。获得对M-RNA剪接的见解 将追求II组内含子的结构自剪接RNA。为了探索替代途径 产生某些古细菌使用的cys-trnacys，phe-tRNA合成酶的古细菌的结构 将用适当的底物结合建立氨基酰化型Trnacys。