The origins of RNA catalysis in ribozymes

The discovery of RNA catalysis provided a paradigm shift in biology, insight into the evolution of life on the planet and a challenge to understand its mechanistic origins. RNA has limited catalytic resources that must be used to maximal effect. Consequently, RNA catalysis tends to be multifactorial, with several processes contributing to an overall significant enhancement of reaction rate. These include general acid-base catalysis, electrostatic effects, and substrate orientation and proximity. The main players are the RNA nucleobases and bound metal ions. Although most ribozymes carry out phosphoryl transfer, the same considerations appear to apply to peptidyl transfer in the ribosome.     

RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II

Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.     

Cleavage of specific sites of RNA by designed ribozymes

Two ribozymes were designed for site-specific cleavage of RNA. A UA site in an undecaribonucleotide was cleaved by a ribozyme consisting of two partially paired oligoribonucleotides with chain lengths of 19 and 15. The other ribozyme, which consists of a 19-mer and a 13-mer, recognized a UC sequence at positions 42 and 43 of 5 S rRNA.     

RNA catalysis: ribozymes, ribosomes, and riboswitches

The catalytic mechanisms employed by RNA are chemically more diverse than initially suspected. Divalent metal ions, nucleobases, ribosyl hydroxyl groups, and even functional groups on metabolic cofactors all contribute to the various strategies employed by RNA enzymes. This catalytic breadth raises intriguing evolutionary questions about how RNA lost its biological role in some cases, but not in others, and what catalytic roles RNA might still be playing in biology.     

RNA self-ligation: from oligonucleotides to full length ribozymes

The RNA-world-theory is one possible explanation of how life on earth has evolved. In this context it is of high interest to search for molecular systems, capable of self-organization into structures with increasing complexity. We have engineered a simple catalytic system in which two short RNA molecules can catalyze their own ligation to form a larger RNA construct. The system is based on the hairpin ribozyme using a 2',3'-cyclophosphate as activated species for ligation. 2',3'-cyclic phosphates can be easily formed and occur in many natural systems, thus being superior candidates for activated building blocks in RNA world scenarios.     

RNA as a catalyst: Natural and designed ribozymes

RNA can catalyse chemical reactions through its ability to fold into complex three-dimensional structures and to specifically bind small molecules and divalent metal ions. The 2′-hydroxyl groups of the ribose moieties contribute to this exceptional reactivity of RNA, compared to DNA. RNA is not only able to catalyse phosphate ester transfer reactions in ribonucleic acids, but can also show aminoacyl esterase activity, and is probably able to promote peptide bond formation. Bearing its potential for functioning both as a genome and as a gene product, RNA is suitable for in vitro evolution experiments enabling the selection of molecules with new properties. The growing repertoire of RNA catalysed reactions will establish RNA as a primordial molecule in the evolution of life.     

RNA polymerase pushing

Molecular motors can exhibit Brownian ratchet or power stroke mechanisms. These mechanistic categories are related to transition state position: An early transition state suggests that chemical energy is stored and then released during the step (stroke) while a late transition state suggests that the release of chemical energy rectifies thermally activated motion that has already occurred (ratchet). Cellular RNA polymerases are thought to be ratchets that can push each other forward to reduce pausing during elongation. Here, by constructing a two-dimensional energy landscape from the individual landscapes of active and backtracked enzymes, we identify a new pushing mechanism which is the result of a saddle trajectory that arises in the two-dimensional energy landscape of interacting enzymes. We show that this mechanism is more effective with an early transition state suggesting that interacting RNAPs might translocate via a power stroke.