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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 action of DNA-relaxing enzyme on H1-DNA complexes was investigated. Complexes of superhelical and relaxed closed circular duplex DNA with H1 were treated with mammalian relaxing enzyme, deproteinized, and electrophoresed on agarose gels. At relatively low ratios of H1 to superhelical DNA, molecules of superhelical density intermediate between those of the starting material and relaxed DNA, the normal product, were generated. At relatively high H1 histone concentrations (H1:DNA greater than 0.4 w/w), the superhelical DNA was not relaxed. Further, no superhelical turns were introduced into relaxed closed duplex DNA at any concentration of H1 tested. Thus, the binding of H1 histone to DNA prevents the action of the relaxing enzyme. Moreover, H1 histone does not appear to unwind the DNA duplex upon binding. The implications of these observations and the previously demonstrated specificity of H1 histone for superhelical DNA are discussed in relation to the structure of chromatin.

Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A7U7 are reported over a concentration range of 6.9 micrometer to 19.6 micrometer in single strand in 1 M NaCl. The rate constants for helix formation are about 2 X 10(6) M-1 s-1 and decrease with increasing temperature yielding an activation enthalpy of -6 kcal/mole. The rate constants for helix dissociation range from 3 to 250 s-1 and increase with increasing temperature yielding an activation enthalpy of +45 kcal/mole. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.

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.