Ribozymes and RNA Catalysis: Introduction and Primer

DAVID M.J. LILLEY AND FRITZ ECKSTEIN

1.1 What are Ribozymes?

Ribozymes are RNA molecules that act as chemical catalysts, a shortening of ribonucleic acid enzymes. In the contemporary biosphere, the known ribozymes carry out a relatively limited range of reactions (Figure 1.1), mostly involving phosphoryl transfer, notably transesterification (the large majority) and hydrolysis reactions. However, the discovery that peptidyl transferase is catalysed by the rRNA component of the large ribosomal subunit significantly extends the range of chemistry to include the condensation of an amine with an sp2-hybridized carbonyl centre. A significantly greater range of chemical reactions may be catalysed by RNA species selected for the purpose, so that ribozymes catalyzing a broader set of reactions may have existed in the past.

1.2 What is the Role of Ribozymes in Cells?

Contemporary ribozymes (Table 1.1) are used for various biological purposes. The nucleolytic ribozymes bring about the site-specific cleavage (or the reverse ligation process) of RNA by attack of a 20-hydroxyl group on the adjacent 30-phosphorus (Figure 1.2A) (Chapters 2–8). This reaction is exploited for the processing of replication intermediates, and in the control of gene expression by metabolite-induced cleavage of mRNA. Ribonuclease P carries out the processing of tRNA in all kingdoms of life, using a hydrolytic reaction (Chapter 9). Several introns are spliced out autocatalytically by ribozyme action, initiated either by the attack of a 20-hydroxyl group located remotely within the intron (group II introns, Figure 1.2B) (Chapter 11), or by an exogenous guanosine molecule (group I introns, Fig 1.2C) (Chapter 10). While "smoking gun" evidence has not yet been found, the similarity of the chemistry of mRNA splicing in the spliceosome to that of the group II introns makes it very likely that this too is RNA catalysed, with the snU4/U6 RNA as the ribozyme (Chapter 13). Lastly, the peptidyl transferase activity of the ribosome catalyses what is arguably the most important reaction of the cell, the condensation of amino acids into polypeptides (Chapter 14). Ribozymes are widespread in nature, from bacteria and their phages, archaea, yeasts and fungi and higher eukaryotes. They are also present in clinically-significant human pathogens such as the hepatitis D virus (Chapter 6). New ribozymes are still being found, both by biochemical approaches and by the bioinformatic analysis of genome sequencing data.

1.3 Ribozymes Bring about Significant Rate Enhancements

Protein enzymes can achieve some extraordinary catalytic rate enhancements. Values of almost 1018-fold are possible, although many generate much smaller accelerations. RNA catalysts tend to produce more modest rate enhancements. For example, the nucleolytic ribozymes typically accelerate their transesterification reactions by around a million-fold relative to the uncatalysed reaction in a dinucleotide, with rates of around 1 min-1. For those ribozymes this may be as fast as it needs to be, since a given site needs to be cut just once. However, while this rate was previously discussed as some kind of speed limit, it appears that this is not an intrinsic limitation, and some redesign of some ribozymes has resulted in very respectable catalytic rates ≥ 10 s-1.

1.4 Why Study Ribozymes?

There are several reasons for studying ribozymes. First, they are active in contemporary living cells, carrying out reactions that are critical for cell viability in some cases; they are therefore legitimate subjects of interest in the complete description of cellular metabolism.

Second, they may have had a key role in the evolution of life on the planet. There is clearly a rather severe "chicken and egg" problem involved in the origins of proteins and translation systems, both of which seem to require the prior existence of the other. Yet in principle a biosphere in which RNA was simultaneously the informational and catalytic macromolecule provides a temporary solution to that problem. Such an RNA world might have existed around 3.5 billion years ago, yet would have been relatively short lived in geological terms, being swiftly replaced by polypeptides that it would have produced. Some of the ribozymes that currently exist, most notably the ribosome perhaps, may be molecular fossils from that time, and therefore their study may offer a partial glimpse of that early metabolism. Although contemporary ribozymes carry out a very limited range of chemistries, selected ribozymes provide an indication of what is achievable by RNA catalysts, and potentially offer a kind of proof-of-principle of an RNA world.

A third reason for studying ribozymes is that they are rather basic biocatalysts, providing a simplified and contrasting perspective on macromolecular catalytic mechanisms compared with enzymes. The last few years have seen significant advances in our understanding of the chemical origins of ribozyme catalysis, and this may cast light on protein-based catalysis in turn.

Lastly, there has been some effort to exploit the potential specificity of ribozymes as therapeutic agents. In principle, the great selectivity of ribozyme-induced cleavage of a chosen sequence could provide an opportunity to interfere with gene expression if targeted to a specific mRNA; this should ideally be the basis for their development into therapeutic drugs. However, this requires that many more problems be overcome, including stability in serum, delivery to the required location of the chosen cell and correct folding into the active conformation in competition with the native folding of the target RNA in vivo. So far only two ribozymes have found their way into...