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 为与DNA或DNA相互作用的蛋白质(和酶)的功能机制建立结构基础 RNA，主要强调大分子组装。特别令人感兴趣的是蛋白质和 参与促进复制、基因表达和重组过程的核酸-中央 分子生物学的教条。进一步阐明蛋白质加工过程中每一步的作用机理 为了合成，我们希望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 包括1)结合了m-RNA、tRNA和延伸因子Tu的70年代核糖体 氨酰-tRNA或延伸因子G，2)Marismortui SOS亚单位与片段的复合体 信号识别颗粒，以及3)70年代核糖体或其SOS亚单位之间的复合体 蛋白分泌和膜蛋白插入通道，转位蛋白。探索两者之间的差异 细菌核糖体及其来自真核生物的更大的对应物，40s亚基，60s亚基的结构 亚基和/或80S核糖体，包括与IRIS RNA形成的起始复合体，并使用核糖体 从酵母或兔网织红细胞中分离出来的将被追踪。探讨抗生素的结构基础 特异性地，形成H.marismortui 50 S亚基的肽转移酶中心的rna将被替换为 对应于真细菌或真核大亚基rRNA的RNA序列及其与 研究了抗生素。为了进一步了解位点特异性重组酶，伽马-三角洲分解酶， 由两个115个碱基对的DNA形成突触复合体并实现重组，整个12个碱基对的结构 亚基与DNA的复合体将用突变的伽马-三角洲分解酶来测定。深入了解m-RNA剪接 第二组内含子自剪接RNA的结构将被追寻。为了探索另一条途径， 产生一些古生菌使用的cys-tRNAys，Phe-tRNA合成酶的古生代同源物的结构 将建立带有磷酸丝氨酸的氨基酰化tRNACys，并结合适当的底物。