Towards the unterstanding of the pre-mRNA splicing reaction: structure and dynamic studies of the 2'-5' AG lariat forming ribozyme and of its complex with catalysis inhibitors

构象开关是控制细胞生命周期的大多数分子过程的基础。细胞剪接机制，剪接体，是一个极其动态的对象，在那里发生几个组装、拆解和催化过程，这些过程都需要蛋白质和核酸构象的特定对接。最近，一个形成核酶的2‘-5’分支被鉴定出来，它经历了一个交换反应，与前mRNA和第二组内含子剪接的第一步有着惊人的相似之处。因此，了解这种小核酶的构效关系有助于理解剪接体催化的机制。在这里，我们建议用溶液中的核磁共振研究套索形成前后2‘-5’AG支链形成核酶的构象和动力学性质。在套索形成之前，在核酶的催化核心观察到了一个显著的缓慢的构象交换过程，这表明很大的灵活性允许RNA调节其活性构象的获得。因此，构象变化预计与催化过程有关，由带正电的离子诱导。本研究的目的是了解核酶催化过程的结构基础。众所周知，带正电荷的小分子与各种RNA靶标相互作用，抑制核酶的催化活性。在这个项目中，我们计划合成新的寡胺，它们作为先导结构的性质既归功于氨基糖苷类抗生素，也归功于氨基环醇。2‘-5’AG形成的核酶与合成程序得到的催化反应最佳抑制剂的络合物将进行结构研究，以揭示催化抑制的机理。这种机制很可能与抑制RNA的构象变化有关，而RNA的构象变化是催化所必需的。

Conformational switches are the basis of most molecular processes that control the cell life cycle. The cellular splicing machinery, the spliceosome, is an extremely dynamic object where several assembly, disassembly and catalytic processes take place, which all require specific docking of the conformations of proteins and nucleic acids. Recently, a 2'-5' branch forming ribozyme has been identified that undergoes a transteriftcation reaction showing striking similarity to the first step of both pre-mRNA and group II introns splicing. Thus, understanding the structureactivity relationship of this small ribozyme can help to understand the mechanism of spliceosomal catalysis.Here we propose to study the conformational and dynamic properties of the 2'-5' AG branch forming ribozyme before and after lariat formation by NMR in solution. A prominent slow conformational exchange process is observed in the catalytic core of this ribozyme before lariat formation, which indicates that a large flexibility allows the RNA to regulate access to its active conformation. Conformational changes are thus expected to be connected with the catalytic process, being induced by positively charged ions. The goal of this study is to understand the structural basis of the catalytic process in this ribozyme.It is well established that small positively charged molecules interact with various RNA targets and inhibit the catalytic activity of ribozymes. In this project we plan to synthesize novel oligoamines, which owe their property as a lead structure to aminoglycoside antibiotics as well as to amino cyclitols. The 2'-5' AG lariat forming ribozyme in complex with the best inhibitor of the catalytic reaction resulting from the synthetic program will be structurally investigated to discover the mechanism of catalysis inhibition. This mechanism is very likely to be connected with the inhibition of a conformational change of the RNA that is essential for catalysis.