Modifications and deletions of helices within the hairpin ribozyme–substrate complex: An active ribozyme lacking helix 1

Abstract

Within the hairpin ribozyme, structural elements required for formation of the active tertiary structure are localized in two independently folding domains, each consisting of an internal loop flanked by helical elements. Here, we present results of a systematic examination of the relationship between the structure of the helical elements and the ability of the RNA to form the catalytically active tertiary structure. Deletions and mutational analyses indicate that helix 1 (H1) in domain A can be entirely eliminated, while segments of helices 2, 3, and 4 can also be deleted. From these results, we derive a new active minimal ribozyme that contains three helical elements, an internal loop, and a terminal loop. A three-dimensional model of this truncated ribozyme was generated using MC-SYM, and confirms that the catalytic core of the minimized construct can adopt a tertiary structure that is very similar to that of the nontruncated version. A new strategy is described to study the functional importance of various residues and chemical groups and to identify specific interdomain interactions. This approach uses two physically separated and truncated domains derived from the minimal motif.

Tuning RNA folding and function through rational design of junction topology

“…For the HCH and HS 6 H junction variants, TOPRNA quantitatively predicts the experimentally measured difference in docking free energy relative to the HHHH junction ( 6 ). Our simulations also corroborate the observation that the ribozyme is tolerant of base pair deletions in the A and B helices ( 42 ), and provides a physical explanation for why helix A but not helix B frays in the 2WJ hairpin ribozyme ( 38 , 39 ). Collectively, this agreement strongly argues that topological constraints imposed by the central junction are a key contributor to the docking stability of the hairpin ribozyme.…”

“…Thus, while a HHHH topology biases the junction towards docking-competent conformations, it is not particularly optimized for orienting the A and B loops in the correct register. Experimental studies have also observed that the hairpin ribozyme is tolerant of shortening the A and B helices ( 42 ).…”