Sprecher
Beschreibung
Early in the evolution of life the replication of genomes and the transcription of functional genes would have been of vital importance. RNA catalysts or ribozymes appear likely to have played a role in such activities. In extant biology, DNA replication is initiated by binding events at an origin of replication that assemble two topologically clamped replication forks capable of sustained polymerization. Similarly, transcription initiates by binding a polymerase to a DNA promoter, followed by a rearrangement into a topologically clamped RNA transcriptional elongation complex. The requirement for initiation of polymerization to be followed by a structural rearrangement into a topologically trapped elongation complex appears quite fundamental to both replication and transcription and we wondered if an RNA polymerase ribozyme could be selected with such properties.
We have selected an RNA polymerase ribozyme that can, just like a DNA dependent RNA polymerase, use a sigma-like specificity primer to locate a promoter sequence. Once found this RNA enzyme rearranges into a topologically clamped form able to stay associated with a single-stranded RNA template. The clamped polymerase stays associated with circular templates but falls off short linear templates indicating that it can move around the template during polymerization. This polymerase deals with randomly selected templates much better than previous ribozymes but still suffers in that its active site does not stay in register with the extending primer found on the template. Future selections may be able to establish this register and enable sustained polymerization.
The polymerase consists of three domains. The first domain was selected for its ability to ligate itself to an RNA primer by phosphodiester chemistry. The second domain was selected to enable the core catalytic core to allow NTP polymerization on a primed RNA template. The third, and most recently selected domain, confers promoter recognition and topological clamping. I will compare the evolution of this artificial ribozyme to that of other naturally existing ribozymes and suggest that such modular evolution was likely to have been common early in the evolution of life. Future in vitro selection may result in a fourth domain that could lead to the emergence of an RNA replicase ribozyme in the laboratory.
