RNA-mediated chemistries: a case of replication and capping

Our current understanding of biology suggests that early life relied predominantly on RNA for both catalysis and the storage of genetic information. Tremendous efforts have been undertaken to confirm that the catalytic abilities of RNA can sustain this “RNA World”. However, the ability of ribozymes to utilize certain chemistries known to be critical for present-day biology, and hence likely to be important for ribo-organisms, has yet to be demonstrated. Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry at phosphorous reaction centres. I have isolated and characterized an RNA-capping ribozyme that retains stereochemistry during the synthesis of a 5¢-5¢ RNA cap. Stereochemical data determined using thio-phosphate modifications, together with an early rate-limiting step, suggest that this ribozyme utilizes two distinct inverting chemical steps - proceeding via a ribozyme-covalent intermediate - during catalysis. A ribo-organism, like contemporary organisms, would have required ribozymes to replicate an RNA genome within some sort of primordial compartment. To provide support for this requirement, I have isolated an improved RNA polymerase ribozyme, referred to as B6.61, from a mutagenized pool containing ~9 ´ 1014 different sequences using a novel large-scale in vitro compartmentalization system. B6.61 polymerized all tested primer-template (PT) complexes faster than its parent, the Round-18 polymerase ribozyme. For one PT complex a rate enhancement of more than 80 fold was observed for extensions longer than one helical turn. The new variant also exhibited improved fidelity on a number of templates. Most interestingly, B6.61 was found to copy one PT complex by almost two complete turns of an RNA helix. To further study RNA-mediated replication, I investigated the role of nucleic acid structure during the course of oligonucleotide extension by T7 RNA polymerase. In addition to normal transcription, the enzyme can produce anomalous transcripts in the absence of a promoter. I have found oligonucleotides that are able to form transient unimolecular loop structures closed by as little as one base-pair to be viable substrates. This intermittent extension process was found to be quite efficient, and adds to the understanding of viral RNA replicative strategies.