Assignment of the hydrogen bonded proton resonances in (Escherichia coli) tRNAGlu by sequential melting

The 300 MHz nuclear magnetic resonance peaks from the hydrogen bonded protons in (Escherichia coli) tRNA(Glu) in Mg(++) solutions have been measured at temperatures between 25 degrees and 74 degrees . Between 25 degrees and 49 degrees four resonances broaden, three of which are assigned to the hU arm and one to a G.C pair of the tertiary structure. By 64 degrees these four resonances have disappeared and the nuclear magnetic resonance spectrum is fitted very well by a computer simulation based upon resonances from the acceptor, T-Psi-C, and anticodon arms. At 66 degrees the resonances from the T-Psi-C arm are lost and at 74 degrees only those from the anticodon are left. All 20 resonances expected from the cloverleaf model have been assigned by comparing the calculated positions of resonances in a particular arm with the stepwise loss of intensity with temperature. The root mean square error between calculated and observed positions is 0.17 ppm. Resonances at the end of helical regions which are sensitive to stacking beyond the helix allow us to conclude that the acceptor arm is stacked upon the T-Psi-C in a regular helix and that G [unk]73 is stacked upon base pair [unk]1-72 but the hU and anticodon arms are not stacked in a regular helix upon the intervening base A [unk]27.