TAILIEUCHUNG - Báo cáo khoa học: Kinetic characterization of the first step of the ribozyme-catalyzed trans excision-splicing reaction

Group I introns catalyze the self-splicing reaction, and their derived ribo-zymes are frequently used as model systems for the study of RNA folding and catalysis, as well as for the development of non-native catalytic reactions. | ễFEBS Journal Kinetic characterization of the first step of the ribozyme-catalyzed trans excision-splicing reaction P. Patrick Dotson II Joy Sinha and Stephen M. Testa Department of Chemistry University of Kentucky Lexington KY USA Keywords group I intron ribozyme RNA self-splicing trans excission-splicing Correspondence S. M. Testa Department of Chemistry University of Kentucky Lexington KY 40506 USA Fax 1 859 323 1069 Tel 1 859 257 7076 E-mail testa@ These authors contributed equally to this work Received 3 March 2008 revised 7 April 2008 accepted 14 April 2008 doi Group I introns catalyze the self-splicing reaction and their derived ribozymes are frequently used as model systems for the study of RNA folding and catalysis as well as for the development of non-native catalytic reactions. Utilizing a group I intron-derived ribozyme from Pneumocystis carinii we previously reported a non-native reaction termed trans excisionsplicing TES . In this reaction an internal segment of RNA is excised from an RNA substrate resulting in the covalent reattachment of the flanking regions. TES proceeds through two consecutive phosphotransesterification reactions which are similar to the reaction steps of self-splicing. One key difference is that TES utilizes the 3 -terminal guanosine of the ribozyme as the first-step nucleophile whereas self-splicing utilizes an exogenous guanosine. To further aid in our understanding of ribozyme reactions a kinetic framework for the first reaction step substrate cleavage was established. The results demonstrate that the substrate binds to the ribozyme at a rate expected for simple helix formation. In addition the rate constant for the first step of the TES reaction is more than one order of magnitude lower than the analogous step in self-splicing. Results also suggest that a conformational change likely similar to that in self-splicing exists between the two reaction steps of TES. Finally multiple .

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