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Kinetic and mechanistic analysis of nonenzymatic, template-directed oligoribonucleotide ligationThe role of divalent cations in the mechanism of pyrophosphate-activated, template-directed oligoribonucleotide ligation has been investigated. The dependence of the reaction rate on Mg2+ concentration suggests a kinetic scheme in which a Mg2+ ion must bind before ligation can proceed. Mn2+, Ca2+, Sr2+, and Ba2+ can also catalyze the reaction. Although Pb2+ and Zn2+ do not catalyze the reaction in the absence of other divalent ions, they significantly modulate the reaction rate when added in the presence of Mg2+, with Pb2+ stimulating the reaction (up to 65-fold) and Zn2+ inhibiting the reaction. The logarithm of the ligation rate increases linearly, with slope of 0.95, as a function of pH, indicating that the reaction involves a single critical deprotonation step. The ligation rates observed with the different divalent metal ion catalysts (Mn2+ > Mg2+ > Ca2+ > Sr2+ = Ba2+) vary inversely with the pKa values of their bound water molecules. The pH profile and these relative ligation rates suggest a mechanism in which a metal-bound hydroxide ion located near the ligation junction promotes catalysis, most likely by deprotonation of the hydroxl nucleophile. The effects of changing either the leaving group or the attacking hydroxyl, together with the large delta S(++) value for oligonucleotide ligation (about -20 eu), are consistent with an associative transition state.
Document ID
20040089772
Acquisition Source
Legacy CDMS
Document Type
Reprint (Version printed in journal)
External Source(s)
Authors
Rohatgi, R.
(Massachusetts General Hospital Boston 02114, United States)
Bartel, D. P.
Szostak, J. W.
Date Acquired
August 21, 2013
Publication Date
April 10, 1996
Publication Information
Publication: Journal of the American Chemical Society
Volume: 118
Issue: 14
ISSN: 0002-7863
Subject Category
Exobiology
Distribution Limits
Public
Copyright
Other
Keywords
Non-NASA Center
NASA Discipline Number 52-20
NASA Discipline Exobiology
NASA Program Exobiology

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