Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV RibozymeReportar como inadecuado

Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV Ribozyme - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

The HDV ribozyme’s folding pathway is, by far, the most complex foldingpathway elucidated to date for a small ribozyme. It includes 6 different stepsthat have been shown to occur before the chemical cleavage. It is likely thatother steps remain to be discovered. One of the most critical of these unknownsteps is the formation of the trans Watson-Crick GU basepair within loop III. The U23 and G28 nucleotides thatform this base pair are perfectly conserved in all natural variants of theHDV ribozyme, and therefore are considered as being part of the signatureof HDV-like ribozymes. Both the formation and the transformation of this basepair have been studied mainly by crystal structure and by molecular dynamicsimulations. In order to obtain physical support for the formation of thisbase pair in solution, a set of experiments, including direct mutagenesis,the site-specific substitution of chemical groups, kinetic studies, chemicalprobing and magnesium-induced cleavage, were performed with the specific goalof characterizing this trans Watson-Crick GU base pair inan antigenomic HDV ribozyme. Both U23 and G28 can besubstituted for nucleotides that likely preserve some of the H-bond interactionspresent before and after the cleavage step. The formation of the more stable transWatson-Crick base pair is shown to be a post-cleavage event, while a possiblyweaker trans Watson-Crick-Hoogsteen interaction seems toform before the cleavage step. The formation of this unusually stable post-cleavagebase pair may act as a driving force on the chemical cleavage by favouringthe formation of a more stable ground state of the product-ribozyme complex.To our knowledge, this represents the first demonstration of a potential stabilisingrole of a post-cleavage conformational switch event in a ribozyme-catalyzedreaction.

Autor: Dominique Lévesque, Cédric Reymond, Jean-Pierre Perreault



Documentos relacionados