Abstract
The mechanism of action of purified wheat germ RNA ligase has been examined. ATP was absolutely required for the ligation of substrates containing 5'-OH or 5'-P and 2',3'-cyclic P or 2'-P termini. Ligation of 1 mol of 5'-P-2',3'-cyclic P-terminated poly(A) was accompanied by the hydrolysis of 1 mol of ATP to 1 mol each of AMP and PPi. Purified RNA ligase catalyzed an ATP-PPi exchange reaction, specific for ATP and dATP, and formed a covalent enzyme-adenylate complex that was detected by autoradiography following incubation with [alpha-32P]ATP and separation of the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein doublet with a molecular weight of approximately 110 kDa, the major product detected by silver staining, was labeled in these reactions. Isolated E-AMP complex was dissociated by the addition of ligatable poly(A), containing 5'-P-2',3'-cyclic P termini, to yield AMP and by the addition of PPi to yield ATP. The unique feature of the reactions leading to an exchange reaction between ATP and PPi and to the formation of an E-AMP complex was their marked stimulation (up to 400-fold) by the addition of RNA. This property distinguishes the wheat germ RNA ligase from other known RNA and DNA ligases which catalyze ATP-PPi exchange reactions and form E-AMP complexes in the absence of substrate. Thus, RNA appears to function in two capacities in the wheat germ system: as a cofactor, to stimulate the reaction of the enzyme with ATP, and as an authentic substrate for ligation.
Highlights
From the *Department of Development Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York10461 and the $Graduate Program inMolecular Biology and Virology, Memorial Sloan-Kettering Cancer Center, New York,New York 10021
A protein doublet with a molecular mini can be ligated by purified wheat germ RNA ligase.The weight of approximately 110 kDa, the major product ligation reaction, which results inthe formation of a 2‘detected by silver staining, was labeled in these reac- phosphomonoester-3’,5‘-phosphodiesterbond is absolutely tions
We have found that the reaction involved in the formation of a covalent complex between wheat germ RNA ligase and AMP is markedly stimulated by RNA, as is an exchange reaction between ATP and PPI
Summary
From the *Department of Development Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York10461 and the $Graduate Program inMolecular Biology and Virology, Memorial Sloan-Kettering Cancer Center, New York,New York 10021. Required for the ligation of substrates containing 5’- Partially purified preparations of wheat germ RNA ligase OH or 5’-Pand 2’,3’-cyclic P or 2‘-P termini. Assays of RNA Ligase and 2‘,3’-Cyclic Phosphodiesterase Actiuities-Reaction mixtures (0.02 ml) containing 20mM Tris-HC1, pH Isolation of E-AMP Complex in the Presence of SDS-Ten individual reaction mixtures, each containing 1 pg of RNA ligase Purified RNA ligasewas unaffected by treatment with micrococcal nuclease but was inactivated by treatment with proteinase K no ligase activity was detectable following extraction with phenol/chloroform (data not shown) These observations indicate that thewheat germ RNA ligaseis a protein that does not contain an RNA component which is essential for the ligation of exogenous substrates.
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