Catalytic Mechanism of Peptide Bond Formation on the Ribosome

研究了核糖体上肽基转移酶（PT）反应的催化机理。核糖体晶体学和核糖体RNA（rRNA）化学修饰的最新结果表明，一般的酸碱催化有助于PT反应，rRNA的某些碱基直接参与催化。然而，迄今为止，缺乏定量数据和催化基团的直接鉴定。本建议的目的是调查的PT反应的动力学机制，通过一个新开发的猝灭流方法，使用肽基-tRNA作为供体和嘌呤霉素作为受体底物。一般酸碱催化对PT反应的贡献将通过测量反应速率的pH依赖性来确定。通过使用具有羟基基团代替氨基基团的嘌呤霉素衍生物，将核糖体的催化基团质子化的影响与亲核试剂的质子化区分开。某些rRNA碱基（A2451，A2447）的假定催化作用将使用含有突变23 S rRNA的核糖体进行动力学研究。最后，将研究肽基-tRNA的氨基酸组成和链长对PT反应速率的影响。

The catalytic mechanism of the peptidyltransferase (PT) reaction on the ribosome will be studied. Recent results from ribosome crystallography and from chemical modification of ribosomal RNA (rRNA) suggest that general acid-base catalysis contributes to the PT reaction and that certain bases of rRNA are directly involved in catalysis. However, quantitative data and the direct identification of catalytic groups are lacking so far. The present proposal aims at investigating the kinetic mechanism of the PT reaction by a newly developed quench-flow method, using peptidyl-tRNA as donor and puromycin as acceptor substrate. The contribution of general acid-base catalysis to the PT reaction will be determined by measuring the pH dependence of the reaction rate. By using a puromycin derivative with a hydroxyl group in place of the amino group, the influence of protonating a catalytic group of the ribosome will be distinguished from the protonation of the nucleophile. The presumed catalytic role of certain rRNA bases (A2451, A2447) will be studied kinetically using ribosomes containing mutant 23S rRNA. Finally, the influence of both amino acid composition and chain length of peptidyl-tRNA on the rate of the PT reaction will be studied.