Mg2+-induced tRNA folding

Mg2+-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attach ed to tRNA, four conformational transitions were revealed when the Mg2+ con centration was gradually increased. The last two transitions were not accom panied by changes in the number of base pairs. The observed transitions wer e attributed to Mg2+ binding to four distinct types of sites. The first two types are strong sites with K-diss Of 4 and 16 muM. The sites of the third and fourth types are weak with a K-diss of 2 and 20 mM. Accordingly, the M g2+-binding sites previously classified as "strong" and "weak" can be furth er subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg2+ binding to a unique strong site selective for Mg2+; bin ding to this site causes only minor A(260) change. The transition at 2 mM M g2+ is accompanied by substantial conformational changes revealed by probin g with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bas es. Fast and slow kinetic phases of tRNA refolding were observed. Time-reso lved monitoring of Mg2+ binding to tRNA suggested that the slow kinetic pha se was caused by a misfolded tRNA structure formed in the absence of Mg2+. Our results suggest that, similarly to large RNAs, Mg2+ induced tRNA foldin g exhibits parallel folding pathways and the existence of kinetically trapp ed intermediates stabilized by Mg2+. A multistep scheme for Mg2+-induced tR NA folding is discussed.