STRUCTURAL STUDIES OF THE 50S RIBOSOMAL SUBUNIT WITH SUBSTRATE ANALOGUES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。Halcarcula marismortuii的50S核糖体亚基为蛋白质合成中的结构-功能关系提供了原子水平的解析。近年来，我们已经解决了大核糖体亚基与几个代表肽基转移酶反应途径不同步骤的小类似物的结构，包括每个单独的底物，一个假定的过渡态中间体，以及反应产物。去年，我们收集了50s晶体的初步数据，这些晶体与同时代表反应结合的两个底物的类似物(在A和P位)络合。已经用RNA底物和经过化学修饰的底物进行了实验，以帮助确定特定原子在肽基转移酶中的作用。我们将获得新的数据来解决50S核糖体亚基的高分辨结构，底物结合在A-和P-位点上，并直接在肽转移之前确定核糖体活性部位的准确构象。此外，我们还获得了在肽基转移酶反应关键位置进行取代的底物。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，并有助于确定核糖体催化能力的来源。除了追求先前解决的过渡态类似物的全RNA版本外，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表明亲核攻击的立体化学。最后，我们将确定50年代的结构，其中最大可能的脱酰化tRNA片段与其E-位结合。这种结构可能使我们了解tRNAs在易位周期中从P-位点传递到E-位点的机制。

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.