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Nucleic Acids Research, Vol 25, Issue 17 3543-3549, Copyright © 1997 by Oxford University Press

ARTICLES

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

AS Sjogren, R Stromberg and BM Sjoberg
Department of Molecular Biology, Stockholm University, S-10691 Stockholm, Sweden.

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 approximately 1 min-1(i.e. for our system k cat/ K m approximately 10(5) 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.
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