STRUCTURAL STUDIES OF THE 50S RIBOSOMAL SUBUNIT WITH SUBSTRATE ANALOGUES

50S 核糖体亚基与底物类似物的结构研究

• 批准号: 7358891
• 负责人: THOMAS Arthur STEITZ
• 金额: $ 0.6万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358891
• 关键词: STRUCTURAL; STUDIES; 50S; RIBOSOMAL; SUBUNIT

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 50S ribosomal subunit from Halocarcula marismortuii has provided atomic level resolution of structure-function relationships in protein synthesis. In recent years, we have solved the structures of the large ribosomal subunit complexed with several small analogues that represent different steps along the peptidyl transferase reaction pathway, including each individual substrate alone, a putative transition state intermediate, and the products of the reaction. Last year, we collected preliminary data on crystals of 50S complexed with analogues representing the two substrates of the reaction bound simultaneously (in A and P-sites). Experiments have been performed with RNA substrates and substrates that have been chemically modified to aid in determining the roles of particular atoms in peptidyl transferase. We shall obtain new data to solve the high resolution structure of the 50S ribosomal subunit with substrates bound in the A- and P- site, to determine the exact conformation of the ribosomal active site directly before peptidyl transfer. In addition, we have obtained substrates with substitutions at positions critical to the peptidyl transferase reaction. Solving the structures of these modified substrates bound to the ribosome will enable us to learn the roles of these groups in peptide bond formation, and help determine the source of the ribosome's catalytic power. In addition to pursuing the all-RNA version of the previously solved transition state analogue, we will also be using chiral intermediate analogues to study the reaction. Determining the structure of a chiral transition state analogue will indicate the stereochemistry of the nucleophillic attack. Lastly, we will determine the structure of the 50S with the largest possible fragment of deacylated tRNA bound to its E-site. This structure may allow us to understand the mechanism by which tRNAs are passed from the P-site to the E-site during the translocation cycle.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。海盐蚌50 S核糖体亚基为蛋白质合成中结构-功能关系提供了原子水平的解析。近年来，我们已经解决了与几个小的类似物，代表不同的步骤沿着肽基转移酶反应途径，包括每个单独的底物，一个假定的过渡态中间体，和反应的产物复合的核糖体大亚基的结构。 去年，我们收集了50 S晶体与类似物复合的初步数据，这些类似物代表了同时结合的反应的两种底物（在A和P位点）。已经用RNA底物和已经化学修饰的底物进行了实验，以帮助确定肽基转移酶中特定原子的作用。我们将获得新的数据，以解决高分辨率结构的50 S核糖体亚基与底物结合在A-和P-网站，以确定确切的构象的核糖体活性位点肽转移前直接。此外，我们已经获得了在肽基转移酶反应的关键位置具有取代的底物。解决这些与核糖体结合的修饰底物的结构将使我们能够了解这些基团在肽键形成中的作用，并帮助确定核糖体催化能力的来源。 除了追求以前解决的过渡态类似物的全RNA版本之外，我们还将使用手性中间体类似物来研究反应。确定手性过渡态类似物的结构将指示亲核攻击的立体化学。 最后，我们将确定50 S的结构，其中脱酰化tRNA的最大可能片段结合到其E位点。这种结构可以让我们理解转运RNA在易位周期中从P位点传递到E位点的机制。