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We have compared the molecular mechanism of thermal unfolding for native tRNA fMet (Escherichia coli) and the denatured species produced by annealing at pH 4.3. Relaxation kinetic measurements reveal that the transitions assigned to melting of TphiC, anticodon, and acceptor stem helices at neutral pH remain essentially unaltered at pH 4.3, but the transition corresponding to coupled melting of tertiary structure and dihydrouridine helix is greatly affected. The Tm of this region is more than 20 degrees higher at pH 4.3 and it has a larger enthalpy formation than in the native state. The transition dynamics are also considerably changed. In contrast to the native structure, tRNA fMet1 and tRNA fMet3 have similar tertiary structure stabilities at pH 4.3. We conclude that the structural difference between native and acid-denatured forms is localized in the tertiary structure-dihydrouridine helix cooperative interaction region of the molecule.

The Mg2+ binding properties of the denaturable tRNAGlu2 from E. coli in 0.1 M Na+, pH7, are characterized by equilibrium dialysis. At 34 degrees C, where the native and denatured conformers are in equilibrium, Mg2+ binding is cooperative. By trapping the tRNA completely in the native conformation at 4 degrees C it is shown that native tRNAGlu2 possesses one strong binding site, K1 = 7.5 x 10(4) M-1 and approximately 36 weak sites with K2 = 8.3 x 10(2) M-1. A significantly lower affinity for the denatured conformer is indicated. We show that Mg2+ effects an allosteric transition from the low affinity denatured conformational state to the high affinity native state and develop the appropriate equations to fit the Mg2+ binding data with physically meaningful parameters. Our results also suggest the previously reported cooperative cation binding to tRNA arises from a cation induced conformational change to the native tRNA conformation and does not reflect the inherent Mg2+ binding properties of the native conformer.

We have examined the 270 MHz nuclear magnetic resonance spectrum and relaxation kinetic behavior of tRNA2Glu (E. coli) in the absence of Mg++, a condition which produces an inactive form of this tRNA. The results show that the denatured form has about five fewer proton resonances in the region from -12 to -15 ppm. Relaxation kinetic measurements reveal that the denatured conformer contains three separately melting helices. The results support a model in which the tertiary structure and dihydrouridine helix characteristic of the native form are unfolded in the denatured state, and are replaced by an altered tertiary structure. The acceptor stem, anticodon, and TpsiC helices are intact in this model for the denatured conformation. The optical changes that accompany melting of the denatured tertiary structure are faster than 10 musec.