Investigation of the base-pairing structure of the anticodon hairpin from E. coli initiator tRNA by high-resolution nmr

The high-resolution nmr spectrum of the anticodon hairpin from E. coli tRNA^fMet has been obtained at a number of different temperatures. The positions of the resonances from interior Watson-Crick base pairs are well accounted for (within 0.1 ppm) by a semi-empirical ring current shift theory, but the terminal base pairs are susceptible to the exact orientation of adjacent bases in single-stranded regions. From a careful examination of the exact way in which resonances disappear at elevated temperatures, we conclude that melting in the nmr experiments occurs when the lifetime of a base pair is reduced to several milliseconds. On the basis of these experiments we are able to assign an nmr T_m to each individual base pair and these should be useful in interpreting the melting behavior of the intact molecule. An “extra” resonance is observed at ～11.3 ppm and, on the basis of its position and temperature sensitivity, it is tentatively assigned to the ring nitrogen proton of a “protected” U residue in the anticodon loop. A strong preference for stacking of a nonbase-paired A residue on an adjacent GC base pair is observed even at temperatures in excess of 52°C.