STUDIES OF POINT MUTANTS DEFINE 3 ESSENTIAL PAIRED NUCLEOTIDES IN THEDOMAIN-5 SUBSTRUCTURE OF A GROUP-II INTRON

Domain 5 (D5) is a highly conserved, largely helical substructure of g roup II introns that is essential for self-splicing, Only three of the 14 base pairs present in most D5 structures (A2 . U33, G3 . U32, and C4 . G31) are nearly invariant, We have studied effects of point mutat ions of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mit ochondria, Though none of the point mutations blocked self-splicing un der one commonly used in vitro reaction condition, the most debilitati ng mutations were at G3 and C4. Following mitochondrial Biolistic tran sformation, it was found that mutations at A2, G3, and C4 blocked resp iratory growth and splicing while mutations at the other sites had lit tle effect on either phenotype, Intra-DS second-site suppressors showe d that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function, At the G3 . U32 wobble pair, the mutant A . U pair blocks splicing, but a revertant of that mutant that can form an A(+). C base pair regains some splicing, A dominant n uclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3. These findings are discussed in terms of the hypothesis of Madhani and Guth rie (H. D. Madhani and C. Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceos omal cognate of D5.