STEREOCHEMICAL SELECTIVITY OF GROUP-II INTRON SPLICING, REVERSE SPLICING, AND HYDROLYSIS REACTIONS

We have previously shown, using phosphorothioate substitutions at spli ce sites, that both transesterification steps of group II intron self- splicing proceed, by stereochemical inversion, with an Sp but not an R p phosphorothioate. Under alternative reaction conditions or with vari ous intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction), In this study, we have d etermined the stereochemical specificities of all of the major model h ydrolytic reactions carried out by the aI5 gamma intron from Saccharom yces cerevisiae mitochondria, For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same a s for the step 1 transesterification reaction. In contrast, the splice d-exon reopening reaction proceeds with an Rp but not an Sp phosphorot hioate at the scissile bond, as does true reverse splicing. Thus, by s tereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing, Finally, a substrate RNA that contains the fi rst exon and nine nucleotides of the intron, when reacted with the int ron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this su bstrate can be cleaved by either the step 1 or the step 2 reaction sit e, Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system a nd permit new studies to be interpreted more rigorously.