Investigation of the thermal unfolding of secondary and tertiary structure in E. coli tRNAfMet by high-resolution Nmr.

Abstract

AbstractThe high‐resolution (300 MHz) proton nmr spectrum of E. coli tRNAfMet has been examined in 0.17M NaCl, with and without Mg2+, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNAfMet, some low field (11–15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure A14–S4U8 base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned to secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mg2+ the tertiary structure base pair is present only to ∼45°C, but in the presence of Mg2+ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A14–S4U8 base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mg2+.