Structural basis for RNA function in ribonucleoprotein complex

蛋白质与RNA结合形成核糖核蛋白，参与高度特异性的遗传信息传递。在这项研究中，我们首先关注tRNA加工和翻译后修饰。我们解析了在每个反应步骤中与tRNA引物复合的添加CCA的酶（Nature，2006）、在三个反应步骤中与tRNA复合的MnmA硫尿苷合成酶（Nature，2006）和与tRNA复合的TillS赖氨酸合成酶<Ile2>（Nature，2009）的晶体结构，并以原子分辨率揭示了它们的动态RNA识别和催化机制。第二，在真核生物和古细菌中，tRNA修饰和激活酶与其同源tRNA形成通道核糖核蛋白复合物，完成复杂的顺序化学反应。我们解决了与tRNAIn复合的tRNA依赖性酰胺转移酶GatDE的晶体结构，以显示复合物如何催化三个连续反应（Science，2006）。我们进一步解决了在真核和古细菌tRNA的位置37处参与yW超修饰合成的5种酶中的3种的晶体结构<Phe>（J.Mol.Biol.，2008 ; Nucleic Acids Res.，2009 ; Proc.Natl.Acad.Sci.USA，2009）。这些研究揭示了每种酶如何以不同的方式利用S-腺苷甲硫氨酸辅因子来实现顺序修饰反应。最后，我们解决了Sec易位子机制的晶体结构以及生物化学分析，其在细胞质中如此合成的蛋白质跨膜易位中起作用（Nature，2009）。因此，我们发现SecYE通道和SecA分子马达相互转换其构象，从封闭到开放的形式，相互激活，以实现蛋白质的移位。

Proteins associate and collaborate with RNAs to form ribonucleoproteins, which act in highly specific genetic information transfer process. In this study, we first focused on tRNA processing and post-translational modification. We solved the crystal structures of CCA-adding enzymes complexed with tRNA primer at each reaction step (Nature, 2006), MnmA thiouridine synthetase complexed with tRNA in three reaction steps (Nature, 2006), and TilS lysidine synthetase complexed with tRNA^<Ile2> (Nature, 2009) and revealed their dynamic RNA recognition and catalytic mechanism at an atomic resolution. Second, in eukarya and archaea, tRNA modifying and activating enzymes form channeling ribonucleoprotein complex with their cognate tRNA to fulfil the complex sequential chemical reactions. We solved the crystal structure of tRNA-dependent amidotransferase GatDE in a complex with tRNAGIn to show how the complex catalyzes three sequential reactions (Science, 2006). We further solved the crystal strictures of 3 out of 5 enzymes involved in synthesis of yW hypermodification in position 37 of eukaryotic and archaeal tRNA^<Phe> (J.Mol.Biol., 2008 ; Nucleic Acids Res., 2009 ; Proc.Natl.Acad.Sci.USA, 2009). These studies revealed how each enzyme utilizes S-adenosyl-methionine cofactor in different way to achieve the sequential modification reaction. Finally, we solved the crystal structure as well as biochemical analysis of Sec translocon machinery, acting in translocation of thus synthesized protein in cytoplasm across the membrane (Nature, 2009). We thus showed that SecYE channel and SecA molecular motor mutually transit their conformation from closed to open form to activate each other to achieve the protein translocation.