Conformational heterogeneity and the determinants of tertiary stabilization in the hammerhead ribozyme from Dolichopoda cave crickets

Abstract

Repetitive DNA elements in Dolichopoda cave cricket genomes contain extended hammerhead ribozymes that are functional in adult crickets, but that exhibit very low self-cleavage activity in vitro relative to other extended hammerhead ribozymes. We find that the parental ribozyme tends to misfold into alternate secondary structures in vitro, complicating analysis of contributions by specific nucleotides to activity under biologically relevant magnesium concentrations. However, minor sequence alterations that stabilize the active secondary structure, without altering candidate tertiary interacting nucleotides, boosted observed rates more than 50-fold (4.4 ± 1.7 min(-1)) and doubled the cleavage extent (>60%) in submillimolar magnesium. Productive alterations included flipping two base pairs in stem I, lengthening stem I and opening stem III to generate a trans-cleaving ribozyme. Specific peripheral nucleotides involved in tertiary stabilization were then identified through kinetic analysis for a series of sequence variants and by correlating plateau cleavage values with band intensity in native gel electrophoresis. These results demonstrate that conformational heterogeneity governs self-cleavage by the wild-type Dolichopoda hammerhead ribozyme in vitro, and they suggest a strategy for improving activity and enhancing the suitability of HHRz for intracellular and biotechnology applications.

Prediction of hammerhead ribozyme intracellular activity with the catalytic core fingerprint

“…A similar observation was also made for other extended hammerhead ribozymes [28]. Such conformational heterogeneity of ribozymesubstrate complexes could explain its low activity in vitro.…”

A mathematical approach for secondary structure analysis can provide an eyehole to the RNA world

“…RNA molecules assume various structures -some of which rather different from the minimum free energy arrangement 7 -depending on relatively unknown conditions. The catalytic activities of any RNA molecule depend on it structure 8 ; hence, different structures of the same sequence perform diverse functions 9 . Moreover, the structure of an RNA molecule provides information about its genetic robustness and evolvability 10 .…”