Nuclear magnetic resonance studies on yeast tRNA^Phe.II. Assignment of the iminoproton resonances of the anticodon and T stem by means of nuclear Overhauser effect experiments at 500 MHz

Resonances of the water exchangeable iminoprotons of the T and anticodon stem of yeast tRNA^Phe were assigned by means of Nuclear Overhauser Effects (NOE's). Together with our previous assignments of iminoproton resonances from the acceptor and D stem (A. Heerschap, C.A.G. Haasnoot and C.W. Hilbers (1982) Nucleic Acids Res. 10, 6981–7000) the present results constitute a complete assignment of all resonances of iminoprotons involved in the secondary structure of yeast tRNA^Phe with a reliability and spectral resolution not reached heretofore. Separate identification of the methylprotons in m⁵C40 and m⁵C49 was also possible due to specific NOE patterns in the lowfield part of the spectrum. Our experiments indicate that in solution the Ψ39 residue in the anticodon stem is orientated in a syn conformation in contrast to the normally observed anti orientation of the uracil base in AU basepairs. Evidence is presented that in solution the acceptor stem is stacked upon the T stem. Furthermore, it turns out that in a similar way the anticodon stem forms a continuous stack with the D stem, but here the $m22G26$ residue is located between the latter two stems (as is found in the X-ray crystal structure). The stacking of these stems is not strictly dependant on the presence of magnesium ions. NOE experiments show that these structural features are preserved when proceeding from a buffer with magnesium ions to a buffer without magnesium ions although differences in chemical shifts and NOE intensities indicate changes in the conformation of the tRNA.