Mg2+-Induced tRNA Folding,

Abstract

Mg²⁺-induced folding of yeast tRNA^Phe was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg²⁺ concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg²⁺ binding to four distinct types of sites. The first two types are strong sites with K_diss of 4 and 16 μM. The sites of the third and fourth types are weak with a K_diss of 2 and 20 mM. Accordingly, the Mg²⁺-binding sites previously classified as “strong” and "weak” can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg²⁺ binding to a unique strong site selective for Mg²⁺; binding to this site causes only minor A₂₆₀ change. The transition at 2 mM Mg²⁺ is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg²⁺ binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg²⁺. Our results suggest that, similarly to large RNAs, Mg²⁺-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg²⁺. A multistep scheme for Mg²⁺-induced tRNA folding is discussed.