Internal Motions of Transfer RNA: A Study of Exchanging Protons by Magnetic Resonance36

Abstract

Proton exchange is a probe of macromolecular structure and kinetics. Its value is enhanced when the exchanging protons can be identified by nmr. After dilution of tRNA-H₂O samples in D₂O, slowly exchanging imino protons are observed, with exchange times ranging from minutes to days. In many cases they originate from the dihydro-uracil region. Most slow exchangers are sensitive to buffer catalysis. Extrapolation to infinite buffer concentration yields the life-time of the closed form, in a two-state model of each base-pair. As predicted by the model, the lifetime obtained by extrapolation is independent of the buffer. Typical lifetimes are 14 minutes for CGI 1 of yeast tRNA^Phe at 17°C, or 5 minutes for U8-A14 of yeast tRNA^Asp at 20°C, without magnesium. For most slow exchangers, magnesium increases the lifetime of the closed form, but moderately, by factors never more than five. The exchange rates of other,fast-exchanging, imino protons, as determined by line-broadening, are found to depend on buffer concentration. Base-pair lifetimes are determined as above. For instance UA6 of yeast tRNA^Phe has a lifetime of 14 ms at 17°C. Base-pairs 4 and 6 have shorter lifetimes than the rest of the acceptor stem. Imidazole is a good catalyst for proton exchange of both the long-and the short-lived base-pairs, whereas phosphate is not. Tris is efficient except for cases where, possibly, access is impeded by its size; magnesium reduces the efficiency of catalysis by tris buffer. From the variation of exchange time vs buffer concentration, one determines the buffer concentration for which the exchange rate from the open state is equal to the closing rate. Remarquably, this concentration takes comparable values for most base-pairs, whether shortlived or long-lived. Buffer effects have also been observed in poly(rA)-poly(rU), for which we derive a lifetime of 2.5 ms at 27°C, and in other polynucleotides. Some of the exchange times identified in the literature as base-pair lifetimes may instead reflect incomplete catalysis.