THE 2 STEPS OF GROUP-II INTRON SELF-SPLICING ARE MECHANISTICALLY DISTINGUISHABLE

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5' and 3' splice sites as well as the deoxyribose substitution of the ribose 2' hydroxyl group at the 5' spl ice site. The data suggest that the two active sites differ in their i nteractions with several of these groups. Specifically, sulfur substit ution of the pro-Sp nonbridging oxygen at the 5' splice site reduces t he chemical rate of the step one branching reaction by at least 250-fo ld, whereas substitution of the pro-Sp oxygen at the 3' splice site ha s only a 4.5-fold effect on the chemical rate of step two. Previous wo rk demonstrated that the Rp phosphorothioate substitutions at both the 5' and 3' splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alteratio ns must be made in a single bifunctional active site to accommodate th e two different reactions.