Expanding the chemical range of RNA-mediated catalysis : structure and mechanism

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All chemical reactions that take place in living cells are catalysed by enzymes, the great majority of which are made of protein. However, a small subset of enzymes are made of RNA, called ribozymes. These are very important for a number of reasons. First, they catalyse some very important biological reactions, such as protein synthesis. Second, they probably played a key role in the early development of life on the planet, when RNA likely served as both the carrier of genetic information and the catalyst for metabolic reactions. Third, understanding how the chemically-simple (compared to combinatorial complexity of protein) RNA can act as a catalyst is a challenge to the biological chemist, and offers general insight into the mechanisms of biocatalysis. Lastly, RNA catalysts could potentially provide a source of useful and novel reagents and tools for organic chemistry, biotechnology and medicine. The great majority of known, natural ribozymes catalyse reactions making or breaking phosphorus-oxygen bonds. How the limited chemical resources of RNA are exploited to catalyse these phosphoryl transfer reactions is understood to some degree, but important questions remain. New ribozymes continue to be found in nature, such as the LINE-1 ribozyme that occurs in a human transposable element. These offer a new perspective on the existing mechanistic classification of ribozymes.A primitive RNA-catalysed metabolism would have required a much greater range of reactions to be accelerated, including 'difficult' reactions such as the formation of carbon-carbon and carbon-nitrogen bonds. In vitro selection provides a source of ribozymes that catalyse different chemical reactions. We have recently solved the crystal structure of a selected RNA that catalyses the transfer of a methyl or alkyl group from O6-methyl guanine or O6-alkyl guanine to a specific nitrogen atom on an adenine nucleotide of the RNA. Methyl transferase ribozymes are currently creating a lot of interest in biological chemistry as the full extent of methylation of RNA and its functions are still being elucidated. We are now using a combination of structural and mechanistic approaches to elucidate fully the mechanism of this ribozyme, and our present information already indicates that the ribozyme uses a sophisticated chemical mechanism to accelerate the reaction. We propose to develop this alkyl transferase ribozyme into a tool to engineer site-specific modification of RNA, including fluorescent probes and sites for crosslinking, first in vitro and subsequently in vivo. Although RNA can carry out remarkable feats of catalysis, with rate acceleration of a million-fold or more, the range of chemistry that is possible will be limited by the relative chemical simplicity of RNA. However the catalytic repertoire of RNA might be greatly expanded by the recruitment of co-enzymes. These are small molecules that bind to enzymes and participate in the reaction that is catalysed. RNA is an excellent receptor for binding small molecules, and this is exploited in biology by riboswitches in mRNA that bind metabolites to control adjacent genes. Many riboswitches bind powerful co-enzymes like S-adenosyl methionine, thiamine pyrophosphate and nicotinamide adenine dinucleotide that could greatly expand the catalytic repertoire of ribozymes, and we have hypothesised that some coenzyme-binding riboswitches have evolved from RNA world ribozymes. We propose to test this idea by reverse-engineering such riboswitches into ribozymes using in vitro selection. Such ribozymes could potentially catalyse a wide variety of chemical conversions including the formation of C-C bonds and oxidation-reduction reactions. Such novel catalysts could have applications in chemical synthesis and diagnostics.