Mutations of the two-nucleotide bulge of D5 of a group II intron block splicing in vitro and in vivo: phenotypes and suppressor mutations.

Domain 5 (D5) is a highly conserved substructure of group II introns that is essential for catalysis of both steps of the splicing pathway. Here we studied the effects of mutations of the conserved 2-nt bulge in the binding and catalytic functions of D5 of intron aI5gamma of yeast mitochondrial DNA. Sequence variants of the 2-nt bulge reduced the rate of self-splicing somewhat. Deletion of one or both bulge nucleotides inhibited splicing more than 10(4)-fold, whereas mutants with a 3-nt bulge were much less debilitated. A novel allele with a symmetrical internal loop in place of the bulge self-splices nearly as actively as the control. Trans-splicing assays of D5 function showed some mutations primarily affect the catalytic function of D5, whereas others chiefly affect its binding function. Representative alleles were transformed into mtDNA and each inhibited splicing and respiratory growth. Pseudo-revertants included suppressor mutations nearby in D5 and one mutant yielded a dominant nuclear suppressor. These experiments provide new evidence that the D5 bulge is crucial for D5 binding and catalysis. It is possible that the bulge bends the D5 helix and that the extent of bending is especially important for D5 binding. The presence and exact nature of the bulge is also likely to have local structural consequences (besides bending) that influence the participation of specific backbone groups in binding and catalysis.