The Hammerhead Ribozyme Structure Brought in Line

Abstract

RNA catalysis seems to be considerably more wide spread than originally thought, with the most prominent example being the ribosome, where RNA catalyses the peptidyl-transferase reaction. Among the most and longest studied catalytic RNAs are the small nucleolytic ribozymes, such as the hairpin, VS, HDV and hammerhead ribozymes. They all catalyse the site-specific cleavage of their own phosphodiester backbone in cis or that of a substrate RNA in trans through a transesterification reaction involving the 2’-OH. A novel crystal structure of the hammerhead ribozyme has just been reported, and this should help to clarify a long-standing debate on the mechanism of catalysis. First identified in the 1980s as a catalytically active element in the replication cycle of certain viroids and the satellite RNA of plant viruses, the hammerhead ribozyme is the smallest naturally occurring RNA endonuclease. The motif has also been found in transcripts from the satellite DNA of amphibians, schistosomes, cave cricket and, most recently, encoded in the genomes of other eukaryotic organisms. The hammerhead ribozyme consists of a catalytic core of 11 conserved nucleotides that are flanked by three helices (Figure 1A). In the absence of divalent metal ions, the structure is extended, but upon addition of Mg , the RNA folds in two well-defined steps into a Y-shaped structure (Figure 1B), as deduced by Lilley and coworkers in studies using comparative gel electrophoresis, FRET, NMR and calorimetry. In this active conformation, a reversible transesterification reaction is catalysed by the hammerhead ribozyme (Scheme 1). 9] During cleavage, the 2’OH of nucleotide C17 is deprotonated and attacks the scissile 3’,5’ phosphodiester bond. Of the two cleavage products one carries a 2’,3’-cyclic phosphate, the other a 5’-hydroxy terminus. In the reverse (ligation) reaction, the 5’-oxygen attacks the cyclic phosphate. For the hammerhead ribozyme, however, the ligation does not proceed as efficiently as seen for the hairpin ribozyme. Both reactions proceed through the same, trigonal-bipyramidal pentacoordinated transition state (Scheme 1), thus meeting the principle of microscopic reversibility. This transition state was deduced from the observation that the chirality of the scissile phosphate, when exchanged for a phosphorothioate, was inverted during the course of the reaction, a hallmark of the SN2 mechanism. In the transition state, the 2’-OH of C17 has to be in line with the adjacent phosphorus and the 5’-oxygen of nucleotide 1.1 (Scheme 1). This requirement and other data detailed below gave rise to presumably the longest-standing debate in the ribozyme field. The first hammerhead ribozyme crystal structures showed a maximal deviation from the ACHTUNGTRENNUNGrequired in-line orientation of the three atoms, at 908. Hammerhead cleavage, however, could be achieved by soaking all RNA crystals with divalent metal ions. While the first observation argued for a ground-state structure to be present in the crystal, the second would indicate that no major rearrangements were necessary to reach the tran[a] R. Przybilski, Dr. C. Hammann AG Molecular Interactions Department of Genetics, University of Kassel Heinrich-Plett-Strasse 40 34132 Kassel (Germany) Fax: (+49)561-804-4800 E-mail : c.hammann@uni-kassel.de Figure 1. The hammerhead ribozyme. A) Secondary structure with stems I, II and III and the 11 conserved nucleotides (bold). Cleavage takes place between nucleotides 17 and 1.1, as indicated by an arrow. Numbers are given according to the conventional scheme. In minimal versions of the ribozyme, either stem I or II is closed by loops (dashed lines). B) Y-shaped conformation of the minimal version of the ribozyme upon addition of magnesium. Naturally occurring ribozymes are endowed with tertiary stabilising structures formed between C) loops L1 and L2 or D) loop L1 and bulge B2 in stem II. The ribozyme format shown in (D) was used for crystallisation.