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.