Hammering out the shape of a ribozyme.

One of the important scientific breakthroughs of the past decade was the discovery that certain RNA molecules have inherent catalytic capacities. These RNAs, or ribozymes, seemed likely to have defined three-dimensional conformations analogous to the structures required for the enzymatic activity of proteins. Since the only RNA structures known at atomic resolution were transfer RNAs and simple duplexes, however, the detailed architecture of an RNA active site was a mystery. Thus, great excitement has been generated by the recently reported crystal structure of a hammerhead ribozyme by David McKay and coworkers at Stanford University [1]. The hammerhead motif is a self-cleaving RNA structure that occurs within the genomes of various plant viroids and virusoids and probably facilitates rolling-circle replic-ation of these infectious agents [2]. The secondary structure of the RNA consists of three base-paired stems extending from a conserved central core region, a layout which crudely resembles the head of a hammer (Fig. 1) [3]. Cleavage occurs through the attack of the 2'-hydroxyl of a specific nucleotide within the core on its adjacent phosphodiester bond to produce 2',3'-cyclic phosphate and 5'-hydroxyl termini. Normally a single-turnover catalyst, the hammerhead has been engineered to be a multiple turnover enzyme by separating the strand containing the cleavage site from the rest of the core [4] (Fig. 1). This design facilitated crystallization by allowing substitution of the substrate strand with an all-DNA strand which binds the ribozyme but is not cleaved. The crystal structure reveals the three-dimensional fold of the hammerhead RNA to resemble a wishbone, with Stems I and II forming the arms and Stem III and the core at the base (Fig. 2) [1]. While the three stems are all A-form helices, the structure of the central core region is created by non-canonical pairings of the phylogenetically conserved nucleotide bases. Despite the defined fold of Fig. 1. Secondary structure of the hammerhead motif. The sub-strate strand is shown in green, the ribozyme strand in red. The cleavage site is indicated by an arrow. the RNA, this is clearly a flexible molecule, as revealed by the existence of three distinct RNA molecules within each asymmetric unit of the crystal. The three molecules, while similar in overall structure, have varying conform-ations of the wishbone arms (Stems I and II). Stems II and III are essentially coaxially stacked, with stacked purine bases of the core in between. The highly conserved sequence CUGA between Stems I …