Several types of RNA have been shown to have catalytic activity directed towards strand cleavage. They were originally observed in the case of ‘self-splicing’ introns, i.e. segments of the immature non-protein-coding mRNA that remove themselves during the formation of mature RNA, as shown in Figure 20a. The term ribozyme has been coined to describe all such catalytic RNA molecules.
Figure 20: (a) Action of a self-splicing ribozyme, removing an intron by self-catalysing the splicing reaction. (Exons are nucleic acid sequences that code for protein; introns are the intervening, non-coding sequences.) A reaction between the C2’ hydroxyl group of ribose in nucleotide A and a target nucleotide in the upstream exon I leads to the breaking of a 5′–3′ phosphoester bond at the end of this exon. One of the cut ends is joined to the ribose C2’ in nucleotide A, closing the intron circle. A reaction between the other cut end and nucleotide A severs the intron tail, leaving the ends of the two exons to be sealed together, (b) A so-called ‘hammerhead’ribozyme engineered for use in direct cleavage of a target mRNA chain. Note how homology between the ribozyme and its target drives this interaction. In this case, cleavage is directed downstream from the translation initiation (start) codon, ensuring that the mRNA is not translated.
Some ribozymes are, as described above, self-splicing, i.e. they catalyse cleavage and resealing of their own nucleotide chain, whereas others catalyse these reactions on separate RNA molecules. The activity of a ribozyme, like that of any macromolecule, depends crucially upon its conformation, and we have already described in the previous section the hairpin loops, alternative folding patterns, and tertiary structure-stabilising devices found in RNA molecules. The catalytic domain of a ribozyme can break and re-form phosphodiester bonds between nucleotides, lowering the activation energy for these reactions just as in protein-catalysed reactions.
Engineered ribozymes that are capable of cleaving specific RNA chains within cells are now used extensively as research tools, as shown in Figure 20b. In this case, a hammerhead ribozyme (so called because of the shape of the predicted RNA structure) has been engineered onto a carrier RNA chain that contains a stretch of sequence complementary to a target mRNA. When base pairing occurs, the catalytic ribozyme core is brought into close proximity to the target mRNA and cleaves it. Such cleavage results in degradation of the mRNA by cellular ribonucleases. It is hoped that such molecules could be used therapeutically to target unwanted gene expression such as by retroviruses (e.g. HIV) or mutated genes. Subsection 4.5 describes two further important applications of nucleic acids as targeting agents for therapeutic or experimental purposes.