Three-Dimensional Structure of the Active Site of Self-Splicing Intervening Sequences

One of the most recent exciting discoveries in molecular biology are catalytic RNAs (ribozymes) that are capable of accelerating phosphodiester cleavage and ligation reactions without involving nucleases of protein nature. From the viewpoint of molecular genetics the ribozymes are selfsplicing introns, or, in other words, intervening sequences (IVS). The self-splicing reaction mediated by the IVS proceeds via two consecutive stages: a) monomer guanosine attacks the 5' splice site to cleave off the 5'-exon, leaving the guanosine covalently attached to the 5'-end of the RNA consisting of the IVS and 3'- exon; b) the free 5'-exon joins to the 3'-exon, completing exon ligation. The a) stage of the reaction is believed to be a conventional Sn2 transesterification which follows the in-line associative pathway only in the presence of divalent cations like Mg 2+ or Mn2+. But many important details of the above pathway are largely unknown (e.g. the identities of the general acid and general base to be necessarily involved in any transesterification process, etc.).

To successfully investigate this problem theoretically in some of its complexity one needs to use methods that are capable of modelling the reaction step as a quantum chemical process. However, the large size of the molecule precludes the use of ab-initio and even semi-empirical methods. The molecular dynamics methods that have shown some degree of success in simulating the properties of biological macromolecules in solution, have force fields that are parametrised far from the region of bond breakage and formation. As a result, their use for studying enzyamtic reactions are theoretically unjustified. One way out is to devise hybrid methods: those which perform quantum calculations for the reaction region and classical dynamics for the rest of the molecule and connect the two in an acceptable manner. Such methods are in the process of implementation in various molecular dynamics packages (and in particular, in CHARMM). So far I have used this method to characterise the transition state and calculate the reaction free energy profile of a small model compound, an acyclic pentacoordinated oxyphosphorane species, in gas phase, in a sphere of water molecule and in the presence and absence of Mg2+ ions. The results more or less corroborate ab initio results in that the solvated dianionic species is seen to have a stable intermediate state while the gas phase results are inconclusive yet.