Site-directed mutagenesis of core sequence elements 9R', 9L, 9R, and 2 in self-splicing Tetrahymena pre-rRNA

The intron within the Tetrahymena thermophila nuclear large rRNA precursor is the best studied example of group I self-splicing introns. In this paper, we examine the structural and functional roles of four internal sequence elements which are characteristic of group I introns in the RNA-catalyzed processing reactions. Oligonucleotide-directed mutagenesis was used to generate mutations in sequence elements 9R', 9L, 9R and 2 of the Tetrahymena intervening sequence. Self-splicing activities of variant precursor RNAs were characterized by in vitro splicing following transcription with T7 or SP6 RNA polymerase. First, we confirm the proposed base pairing of sequence elements 9R and 9R' by construction and analysis of compensatory mutations. Mutations in elements 9R (G272A C274G) and 9R' (G100C C102U) each disrupt the pairing and eliminate self-splicing activity. A compensatory 9R/9R' mutation (G100C C102U G272A C274G) restores pairing and normal splicing activity. We conclude that 9R X 9R' pairing is a requirement for self-splicing. Second, we show that self-splicing activity is very sensitive to both nucleotide sequence and RNA secondary structure in the pairing segments of elements 9L and 2. Mutations within these regions at positions 266, 268, 307, and 309 can increase as well as decrease activity relative to wild type. Third, a mutation in the highly conserved nonpairing segment of element 9L (U259A A261C) increases KM for GTP from 29 to 120 microM, but does not otherwise affect splicing activity. The primary consequence of this mutation is a decrease in GTP binding energy of approximately 0.9 kcal/mol. Last, we show that a mutation in the highly conserved nonpairing segment of element 2 (A301C A302G G303C) eliminates transesterification activity, but does not affect 3' splice site hydrolysis.