Proton nuclear magnetic resonance of modified bases of valine transfer ribonucleic acid (Escherichia coli). Direct monitor of sequential thermal unfolding

Abstract

Proton magnetic resonances at 220 MHz from three nucleotide residues of valine I tRNA (Escherichia coli) serve as intrinsic probes of local molecular structure. Resonances from the methyl group of ribothymidine, the methyl group of N6-methyladenosine, and the C-5 methylene of dihydrouridine monitor separate conformational transitions in the TpsiC, anticodon, and dihydrouridine loops, respectively. As the temperature is raised in a solution containing 0.23 M Na+ and no Mg2+, the dihydrouridine region melts with a Tm of 55 degrees, the anticodon region at 58 degrees, and the TpsiC region at 67 degrees. The dihydrouridine nuclear magnetic resonance (NMR) transition correlates with the major change in absorbance monitored in the uv at 330 nm which is ascribed to structural pertubations near the 4-thiouracil moiety. On the NMR time scale slow exchange is seen throughout the temperature range for dihydrouridine and below the apparent Tm for the ribothymidine methyl group. Chemical shift and line width differences between folded and unfolded forms of the polynucleotide indicate that, in the native structure, ribothymidine is in a highly structured region and N6-methyladenosine is in a somewhat less restricted environment. Narrow line widths for the C-5 methylene triplet are found over the whole temperature range indicating that this base is undergoing rapid internal reorientation relative to the overall macromolecule.