The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA

Abstract

The molecular mechanism of thermal unfolding of Escherichia coli tRNA fMet (in 0.17 m-NaCl without Mg 2+) has been elucidated by a combination of relaxation kinetics and proton nuclear magnetic resonance spectroscopy. We measured the n.m.r. ‡ ‡ n.m.r., nuclear magnetic resonance; hUra, dihydrouracil. spectrum of the hydrogen-bonded ring NH protons at different temperatures and found that the resonances assigned to each arm of the cloverleaf broaden and disappear together, yielding four distinct n.m.r. “melting” transitions. Temperature-jump measurements in the same solvent showed five co-operative melting transitions, varying in relaxation time from a few microseconds to ten milliseconds. The relaxation and n.m.r. measurements were correlated by the following model. When the lifetime of a hydrogen-bonded proton in a helix is five milliseconds, its n.m.r. line will be broadened to approximately twice its intrinsic low-temperature width and appear to “melt”. The helix dissociation time constants of the relaxation effects were extrapolated by the Arrhenius equation to lower temperatures where their values were five milliseconds. The correlation of extrapolated dissociation time constants with n.m.r. melting of specific helices allowed assignments of the structural basis for each relaxation effect. The results show that the principal path for the reversible thermal unfolding of tRNA 1 fMet under these solution conditions is first, transient opening of the dihydrouridine helix, followed by simultaneous melting of the dihydrouridine helix and a “tertiary” interaction, which does not correspond to a cloverleaf helix. The tertiary interaction is much less stable in tRNA 3 fMet, with T m lowered by 16 °C from tRNA 1 fMet. The sequence of melting steps at higher temperatures is the same in the two isoacceptors: first the TΨC helix melts, followed by the anticodon helix and finally the acceptor stem helix. Thermodynamic and kinetic parameters are reported for these steps. The method of sequential melting, combining n.m.r. and relaxation kinetic techniques, is a powerful procedure for elucidating RNA secondary structure. In addition, this method allows assignment of many hydrogen-bonded ring NH proton resonances that are unresolved in the low-temperature spectrum.