EXON STRUCTURE AND SPLICING OF GROUP I INTRONS

Johns Hopkins University, Baltimore MD

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Abstract

DESCRIPTION: RNA processing is intimately related to its structure, as RNA interactions organize the catalytic center of processing complexes and create or mask sites for regulatory proteins. The goal of the proposed research is to understand how the long-range folding of RNA influences gene expression using the Tetrahymena self-splicing group I intron as a model system. This system is advantageous for studying RNA structure because the catalytic activity of the intron directly reflects its conformation. Alternative secondary structures attenuate self-splicing by trapping the pre-ribosomal RNA in an inactive conformation during transcription in vitro. In vivo, kinetic barriers to splicing are overcome by factors not yet understood. RNA interactions that lead to the formation of inactive or active pre-rRNA will be identified by chemical modification and phosphorothioate interference. The effect of mutations, temperature, magnesium concentration, and the transcription process itself on the competition between inactive and active RNA structures will be determined. Direct strand-exchange between alternative helices in inactive and active pre-rRNA will be tested by disrupting stacking of adjacent helices and by trapping unpaired sequences with complementary DNA oligonucleotides. These experiments will establish a general framework for RNA folding kinetics that is expected to be relevant to other systems such as the spliceosome. Factors that facilitate RNA folding in vivo will be investigated using bacterial genetics. The Tetrahymena intron is spliced as rapidly from the homologous position of 23S rRNA in E. coli as it is in Tetrahymena. Ribosomal RNA sequences will be randomly mutated and selected for inhibition of splicing. Proteins that facilitate folding of splicing-competent pre-rRNA will be identified by complementation of splicing-defective pre-rRNA. This work will be directly relevant to pre-rRNA processing, splice site selection, and intron evolution.