Key result
Sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduced the chemical rate of step one branching by at least 250-fold, whereas substitution at the 3' splice site had only a 4.5-fold effect on step two.
Effect estimate: 250-fold reduction (step one), 4.5-fold effect (step two)
The two steps of group II intron self-splicing are mechanistically distinguishable, suggesting either two distinct active sites or significant alterations in a single bifunctional active site.
Distinguishes the two splicing steps mechanistically; leaves open whether single or dual active sites operate in human spliceosomes.
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' splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3' splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work 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 alterations must be made in a single bifunctional active site to accommodate the two different reactions.
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Podar et al. (1998) studied this question. Sulfur substitution of nonbridging oxygens at 5' and 3' splice sites vs. Unsubstituted introns was evaluated on Chemical rate of the step one branching reaction and step two reaction (250-fold reduction (step one), 4.5-fold effect (step two)). Sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduced the chemical rate of step one branching by at least 250-fold, whereas substitution at the 3' splice site had only a 4.5-fold effect on step two.