Experimental simulation on molecular evolution of RNA enzymes (ribozymes)

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We have been investigating the structure-function relationship of the Tetrahymena ribozyme that is the most extensively studied group I self-splicing intron. During the course of this project, two new stem-loop structures (P2.1 and P9.1) in the RNA are identified as the peripherals that associate together via long-range Watson-Crick basepairings. It also became clear that one of them functions as a trans-activator for the intron lacking this region. The activation presumably depends on an unusual A-C baseparing and the nucleotides located nearby.A new technique by that active RNAs can be selected among the modified ones with nemerous numbers of mutations was developed. The technique is based on the combined use of in vitro selection and color colony assay. By employig this technique, we identified adenosines that are located close to each other in an unusual loop region are resposible for the activation of the intron RNA.A project that employs error-prone PCR technique is also now in progress.On the basis of these findings, we are now attempting to build new artificial domains that facilitates tthe splicing reaction by the catalytic RNA.