Efficient self-splicing of the Tetrahymena group I intron depends on its exon context. The objective of this proposal is to elucidate the mechanism of the 5' splice site recognition in natural precursors. In the first step of splicing, G-addition at the 5' splice site, depends on base pairing between the 5' exon and the guide sequence of the intron. In natural precursors, an alternative hairpin potentially inhibits recognition of the 5' splice site and reduces the rate of splicing. This depends on the length of the exons: precursors with long exons splice 20-fold more rapidly than precursors with short exons. Current data indicate that 120 nucleotides 5' and 70 nucleotides 3' exon of the intron are needed. The inhibitory hairpin is a conserved feature of rRNA. Thus, these preliminary data imply that a conformational change occurs in the rRNA upon splicing, and that this change is required for efficient excision of the intron. The exon requirements for efficient splicing will be determined by site-directed and random mutagenesis. The equilibrium between active and inactive conformations of the precursor will be assayed using hybridization of RNA or DNA oligomers to targeted """"""""single-stranded"""""""" portions of the precursor transcript. This equilibrium may be affected by tertiary interactions with other portions of the transcript. In particular, a conserved GU pair in the inhibitory hairpin may be responsible for interactions with the intron core. This GU pair will be changed to an AU. The secondary structure of the exon sequences will be determined using chemical modifying agents and single-strand specific riboendonucleases. Folding of the exon sequences in the precursor and the spliced transcript will be compared. Alternative secondary structures predicted using computational methods will be tested by site-specific mutagenesis. Emphasis will be placed on identifying changes in the exon sequences that occur during splicing. Splicing of the Tetrahymena intron from the E. coli rRNA will be tested in vitro and in vivo. The intron will be imbedded in the E. coli large rRNA sequence at the position analogous to its location in the Tetrahymena sequence. The potential to adopt secondary structures which obscure the 5' splice site and inhibit splicing is conserved between prokaryotic and eukaryotic rRNAs. It is not known whether the E. coli rRNA also adopts a conformation that permits excision of a group I intron.
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