J. Biol. Chem., Vol. 267, Issue 4, 2429-2436, Feb, 1992
Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis
D Smith, AB Burgin, ES Haas and NR Pace
Department of Biology, Indiana University, Bloomington 47405.
A high yield, photoactivated cross-linking reaction between a modified tRNA
and RNase P RNA was used as a quantitative assay of substrate binding
affinity. The cross-linking assay allows the effects of metal ions on
substrate binding to be measured independently and in the absence of the
pre-tRNA cleavage reaction. The results of this assay, in conjunction with
the conventional cleavage assay, support the following conclusions about
the nature of the RNase P RNA-tRNA binding interaction. (i) Monovalent
cations act primarily to enhance enzyme- substrate binding, presumably by
functioning as counterions. This enhancement can be attributed to a
reduction in the tRNA off-rate. (ii) Although divalent cation is required
for cleavage, the enzyme-substrate complex can form in the absence of
divalent cation; the essential role of divalent cation in the reaction is
thus catalytic. (iii) Ca2+ is as efficient as Mg2+ in promoting binding but
supports catalysis only at a low rate.
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