Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis.
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.
Smith
D
D
Department of Biology, Indiana University, Bloomington 47405.
Burgin
A B
AB
Haas
E S
ES
Pace
N R
NR
D001345:Autoradiography; D002384:Catalysis; D002413:Cations, Divalent; D002414:Cations, Monovalent; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D010957:Plasmids; D011073:Polyamines / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D043262:Ribonuclease P; D013379:Substrate Specificity
D002413:Cations, Divalent; D002414:Cations, Monovalent; D003432:Cross-Linking Reagents; D008670:Metals; D011073:Polyamines; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P