Tritium exchange on transfer RNA: slowly exchanging protons sensitive to a change in the dihydrouridine stem.

Abstract

Measurements of tritium exchange on tRNA were made for periods from 0.5 min to 8 hr after separation from labeled solvent. The exchange curve was analysed in terms of three kinetic classes of exchanging protons with half-lives of 5 hr (12 protons), 0.54 hr (37 protons and about 3.5 min (58 protons) at 00C in 0.14 M K+/10 mM Mge. The behaviour under varying ionic conditions of protons in the slowest exchange class and of some protons in the intermediate class suggests that they are dependent on the tertiary structure of the molecule. Moreover, in the same range of exchange times characteristic of these latter protons, about 9 more protons were observed in the case of a mutant form of tRNATrP, the UGA-suppressor species, than in the wild-type tRNATrP. These two species differ only in base 24 in the dihydrouridine stem. This dynamic difference between the wildtype and suppressor species may be related to a functionally important difference in coupling between the conformation of the molecule and interactions at the anticodon.ture of tRNA and suggest that tertiary structure contributes an additional source of slowly exchanging hydrogens (6, 7, 12).There is little information available to account for the overall kinetic behaviour of the protons within tRNA structure that exchange slowly. We describe here protons exchanging with a half-life of the order of hours, observable in the presence of Mg2e or spermine or 1.4 M KC1 but not in 0.14 M KCI alone.This suggests that these protons owe their slow exchange to the integrity of the tRNA tertiary structure. Furthermore, the number of these protons is sensitive to a base change in the D stem in the case of tRNATrP (Escherichia coli). We have analyzed the overall behavior of the slowly exchanging protons in terms of three classes, with average half-lives of 5 hr, 0.5 hr, and 3.5 min. We describe the behavior of these three classes under different ionic conditions and discuss their origin.During the course of its biological function, a tRNA molecule interacts with many different macromolecular species (1). Many authors have emphasized the essentially dynamic molecular structure that tRNA must have to account for these diverse interactions (2-4). Indeed, the isolated molecule in solution is conformationally heterogeneous and strongly affected by ionic conditions, such as the presence of monovalent and divalent cations and polyamines (3-7). One particularly useful probe of the dynamic aspects of nucleic acid structure is the tritium-exchange technique largely developed by Englander et aL (8). It has been shown with this technique that in double helices, five protons associated with G-C base pairs (bp) and three protons in A-U bp exchange slowly enough to be labeled with tritium and measured by Sephadex-column or fast-dialysis techniques (9, 10). All except the guanine exocyclic amino protons are dependent on transitory opening of the bp for exchange.Models favored for the opening process have usually supposed that there is a cooperative opening of several bp in ord...

Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance

“…Recently, Ramstein & Buckingham (1980) have presented new tritium exchange data for tRNA. They find that several protons in unfractionated E. coli tRNA exchange in several hours and have also observed an interesting difference in exchange behavior between E. coli tRNATrp and the UGA suppressor which differs in one base pair from it in the D stem.…”

Biomolecular dynamics: A report from a workshop in Gysinge, Sweden, October 4–7, 1982

“…They show that there exist 3 different classes of protons with half-lifes of 3 min, 32 min and 5 h. Changing the ionic strength such that this leads to a stabilization of the tertiary structure also alters the population in the classes. From this one can deduce that the slowly exchanging protons and some of the intermediate ones are involved in or affected by tertiary interactions (Ramstein & Buckingham, 1981).…”