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

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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.