核糖体RNA甲基转移酶催化不同类型底物 (RNA/核糖体亚基)的碱基甲基化分子机理研究

核糖体RNA的甲基化对核糖体的生物合成和活性具有重要的调节作用，其中碱基甲基化是常见的修饰类型，与细胞内蛋白表达调控以及细菌耐药性密切相关。RNA甲基转移酶底物特异性的明显差异以及修饰核苷酸位点的不同决定了它们具有不同的底物识别机制。本研究选取不同底物识别类型的三种甲基转移酶RlmG(m2G1835)、RsmH(m4C1402)和RsmE(m3U1498)作为研究对象，它们作用于核糖体装配的不同阶段，利用蛋白质晶体学方法解析它们与底物 (RNA或核糖体亚基)及甲基供体的三元复合物晶体结构。在此基础上，将深入探索蛋白突变体在体内和体外的甲基转移酶活性及与底物的相互作用，在核糖体水平上系统的阐明作用于核糖体装配不同阶段的RNA甲基转移酶识别底物碱基上特异位点的机理,以及这些修饰如何影响蛋白合成等重要生理过程。目前我们已解析了这三种酶的高分辨率晶体结构，为研究复合物结构与功能奠定了基础。

Methylation of ribosomal RNA (rRNA) nucleotides plays an important role in the biogenesis and regulation of the ribosome in all living organisms. Bases methylation of rRNA is a common type of modification closely associated with fine-tuning the process of protein synthesis and bacterial resistance to antibiotics. There are distinct substrate patterns in different RNA methyltransferases (MTases): some MTases can only recognize ribosome subunits while some can only protein-free RNA. This difference most likely result from their different structures and different modified nucleotides, and thus brings about different catalyzing mechanisms, although there were a few studies on the structures of MTases in complex with their substrate RNA. In this research, the MTases RlmG (m2G1835), RsmH (m4C1402) and RsmE (m3U1498) are applied as our objects of study, as they catalyze the rRNA methylation from different types of substrates during different stages of ribosome assembly, and we plan to solve their crystal structures in complex with the substrates (RNA or ribosome subunits) and AdoMet. On this basis, the in vivo and in vitro MTase activities of a series of mutants and their interactions with the substrates will be further studied.This study will systematically demonstrate the detail mechanisms of base methylation in different nucleotides of rRNA catalyzed by the MTases with different substrate-recognizing patterns, and its implications for some important physiological processes such as protein synthesis and resistance. Now, we have solved the high resolution structures of the three MTases, providing the basis for further studies on the structures and function of their complexes.