In vitro oligonucleotide-directed mutagenesis, transcription and RNA splicing analysis will be used to study the self-catalyzed splicing of the intervening sequence (IVS) within the precursor of the Tetrahymena thermophila 26S ribosomal RNA. The goals of the project are: first, to experimentally determine whether conserved intron sequences and/or potential secondary interactions are involved in catalysis of the RNA splicing reactions; second, to identify the specific transesterification and hydrolysis step(s) in processing which are affected by each nucleotide sequence alteration; and, third, to integrate these results with the results of others who are studying the structure and function of this intron and other Group 1 introns with the eventual goal of elucidating the biochemical mechanism of RNA-catalyzed splicing. We will generate single mutations and compensating double mutations within conserved sequence elements 9L, 2, 9R, 9R', A and B in recombinant plasmid pSPTT1A3, which contains the intron and attenuated exon sequences cloned downstream of the SP6 transcriptional promoter. Transcription and processing analysis of mutant and wild type precursor RNA molecules will be carried out in vitro. This project is being carried out in collaboration with Dr. Thomas Cech and coworkers (University of Colorado). Results will be of broad biological significance, since the experiments bear directly on the mechanism of RNA splicing, catalysis by RNA, the structure of RNA, and the evolution of split genes.
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