Poster abstracts

Poster number 62 submitted by Dan Kellerman

Testing a proposed base-pairing interaction between the enzyme and substrate in the hepatitis delta virus (HDV) ribozyme using multiple alternative substrate kinetics

Daniel L. Kellerman (Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Kandice Simmons (Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Mayra Pedraza (Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Darrin M. York (Department of Chemistry and Chemical Biology, BioMaPS Institute for Quantitative Biology, Rutgers, The State University of New Jersey), Michael E. Harris (Department of Biochemistry, Case Western Reserve University, Cleveland, OH )

Abstract:
The hepatitis delta virus (HDV) is a small, satellite RNA virus of the hepatitis B virus. HDV infection results in severe acute hepatitis with a high risk of chronic liver disease progressing to cirrhosis. Replication of HDV requires the activity of the HDV ribozyme (HDVrz), a small, catalytic RNA motif that undergoes a self-cleavage reaction to produce viral unit-length genomes. Understanding how the HDVrz catalyzes self-cleavage is important for understanding mechanisms of viral pathogenesis and provides important insights into biological catalysis. We have developed methods for measuring the relative catalytic activity of multiple alternative substrates in a single reaction by quantifying the ratios of substrate concentrations as a function of reaction progress using an internal competition assay. Here, we apply this multiple alternative substrate kinetic approach to the HDVrz, in order to provide a systematic analysis of proposed interactions between the substrate and HDVrz. Previous molecular mechanics simulations of the HDVrz reaction indicated the potential for formation of a Watson-Crick base pair between the catalytic RNA and its substrate that is necessary in positioning the nucleophile for catalysis. The modeling implicates that A79 in the J 4/2 region of the ribozyme forms a canonical Watson-Crick base pairing interaction with the U(-1) on the substrate. To test this hypothesis we created mutant enzymes and substrates with all four nucleotide possibilities at each position. Disruption of the base pair between A79:U(-1) should decrease catalytic activity, while compensatory mutations that restore base paring would be expected to rescue activity. Additionally, as the nucleobase identity of the (-1) position on the substrate strongly influences HDVrz catalytic activity, we have created substrate mutants containing nucleotide analog substitutions to provide further insight into the specific interactions of the (-1) position with the ribozyme.

Keywords: catalytic RNA, HDV ribozyme