Determination of hepatitis delta virus ribozyme N(-1) nucleobase and functional group specificity using internal competition kinetics.

Autor: Kellerman DL; Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA., Simmons KS; Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA., Pedraza M; Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA., Piccirilli JA; Department of Chemistry and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA., York DM; Center for Integrative Proteomics Research, BioMaPS Institute for Quantitative Biology and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA., Harris ME; Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Electronic address: meh2@cwru.edu.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2015 Aug 15; Vol. 483, pp. 12-20. Date of Electronic Publication: 2015 May 01.
DOI: 10.1016/j.ab.2015.04.024
Abstrakt: Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2'-O-transphosphorylation by the hepatitis delta virus (HDV) ribozyme is sensitive to the identity of the N(-1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2'O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(-1) nucleobase contributes to HDV catalysis and test the utility of this approach for RNA structure-function studies. Internal competition reactions containing all four substrate sequence variants at the N(-1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed base-pairing interaction. Mutants A78U, A78G, and A79G retain significant catalytic activity but do not alter the specificity for the N(-1) nucleobase. Effects of nucleobase analog substitutions at N(-1) indicate that U is preferred due to the ability to donate an H-bond in the Watson-Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site and illustrate multiple substrate kinetic analyses as a practical approach for characterizing structure-function relationships in RNA reactions.
(Published by Elsevier Inc.)
Databáze: MEDLINE
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