LENGTH CHANGES IN THE JOINING SEGMENT BETWEEN DOMAIN-5 AND DOMAIN-6 OF A GROUP-II INTRON INHIBIT SELF-SPLICING AND ALTER 3'-SPLICE-SITE SELECTION

Domain 5 (D5) and domain 6 (DG) are adjacent folded hairpin substructu res of self-splicing group II introns that appear to interact within t he active ribozyme. Here we describe the effects of changing the lengt h of the 3-nucleotide segment joining D5 to D6 [called J(56)3] on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitocho ndrial DNA. Shortened variants J(56)0 and J(56)1 were defective in vit ro for branching, and the second splicing step was performed inefficie ntly and inaccurately, The lengthened variant J(56)5 had a milder defe ct-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice. Yeast mitochondria mere t ransformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing. Respi ration-competent revertants were recovered, and in one type a single j oiner nucleotide was deleted while in the other type a nucleotide of D 6 was deleted, Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the se cond step of splicing, both spliced accurately in vivo. These results establish that a 3-nucleotide 5(56) is optimal for this intron, especi ally for the accuracy of 3' splice junction selection, and indicate th at D5 and D6 are probably not coaxially stacked.