Magnesium ions added to tRNAfMET1 selectively stabilize the dihydrouridine helix-tertiary structural region. Low Mg2+ levels have little direct effect on the remaining three cloverleaf helices, but these are prevented from melting independently when their intrinsic Tm is surpassed by the Tm of the tertiary structure. At high Mg2+ concentration the thermal unfolding of tRNAfMet1 is approximately a two-state, concerted transition from the globular native structure to the random coil, in contrast to the sequential unfolding observed without Mg2+. We interpret the kinetics of refolding to mean that the D helix serves as a required nucleus for the rate-limiting step of tertiary structure formation. We found that unfolding of the tertiary structure leads to loss of the tightly bound Mg2+ ions, and showed with a Mn2+-sensitive fluorescent indicator that the rate of Mn2+ release is the same as the rate of unfolding the tertiary structure. Hence the tightly bound divalent ion must be located in a site formed by the tertiary structure-D helix region of the molecule.
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