pH dependence of self-splicing by the group IA2 intron in a pre-mRNA derived from the nrdB gene of bacteriophage T4

Abstract

The nrdB gene of bacteriophage T4 contains a group IA2 intron. We have investigated the kinetics of self-splicing by a shortened variant of nrdB pre-mRNA in the presence of the co-substrates guanosine and 2'-amino-2'-deoxyguanosine. The pH dependence of the first transesterification step displayed parallel linear correlations for the two different co-substrates up to pH 7, above which the reaction with guanosine levels off to become pH independent. The plot for the 30-fold slower reaction with 2'-aminoguanosine is linear up to pH 8-8.5 and then levels off. The linear correlations with slopes close to unity suggest that a deprotonation event accelerates the transesterification reaction and that a change in rate limiting step occurs at a first order rate constant of ~ 1 min-1 (i.e. for our system k(cat)/K(m) ≃ 105 M-1 min-1). The pH dependence of observed rate constants in different divalent metal ion mixtures, where the 2'-aminoguanosine-dependent reaction is enhanced 6- and 35-fold compared with that in magnesium, strongly supports this conclusion. This is, to our knowledge, the first report on an intact self-splicing group I intron where use of different co-substrates and divalent metal ions shows that a deprotonation enhances the rate and verifies that the transitions occurring during splicing of group I introns are all part of a common reaction sequence.