Mutational inhibition of ligation in the hairpin ribozyme: substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage.

ABSTRACT: The hairpin ribozyme acts as a reversible, site-specific endoribonuclease that ligates much more rapidly than it cleaves cognate substrate. While the reaction pathway for ligation is the reversal of cleavage, little is known about the atomic and electrostatic details of the two processes. Here, we report the functional consequences of molecular substitutions of A9 and A10, two highly conserved nucleobases located adjacent to the hairpin ribozyme active site, using G, C, U, 2-aminopurine, 2,6-diaminopurine, purine, and inosine. Cleavage and ligation kinetics were analyzed, tertiary folding was monitored by hydroxyl radical footprinting, and interdomain docking was studied by native gel electrophoresis. We determined that nucleobase substitutions that exhibit significant levels of interferen

SUBMITTER: Gaur S 

PROVIDER: S-EPMC2151026 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature