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

构象转换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，是一个非常动态的对象，其中发生几个组装，拆卸和催化过程，这都需要蛋白质和核酸的构象的特定对接。最近，已经鉴定了形成2 '-5'分支的核酶，其经历的转酯化反应显示出与前体mRNA和II组内含子剪接的第一步惊人的相似性。因此，了解这种小核酶的结构-反应性关系有助于理解剪接体催化机制，本文拟用核磁共振技术研究核酶形成2 '-5' AG分支前后的构象和动力学性质。一个突出的缓慢的构象交换过程中观察到这种核酶的催化核心前lactinase形成，这表明，一个大的灵活性允许RNA调节其活性构象的访问。因此，预期构象变化与催化过程有关，由带正电荷的离子诱导。本研究的目的是了解这种核酶催化过程的结构基础。众所周知，带正电的小分子与各种RNA靶点相互作用并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，这归功于他们的财产作为一个领先的结构，以氨基糖苷类抗生素以及氨基环醇。本文将从结构上研究由合成程序得到的与催化反应的最佳抑制剂复合的2 ′-5 ′ AG酶形成核酶，以发现催化抑制的机理。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.