Single-molecule FRET reveals a cooperative effect of two methyl group modifications in the folding of human mitochondrial tRNA(Lys).

Using a combination of advanced RNA synthesis techniques and single molecule spectroscopy, the deconvolution of individual contributions of posttranscriptional modifications to the overall folding and stabilization of human mitochondrial tRNA(Lys) is described. An unexpected destabilizing effect of two pseudouridines on the native tRNA folding was evidenced. Furthermore, the presence of m(2)G10 alone does not facilitate the folding of tRNA(Lys), but a stabilization of the biologically functional cloverleaf shape in conjunction with the principal stabilizing component m(1)A9 exceeds the contribution of m(1)A alone.

Single-molecule FRET studies of counterion effects on the free energy landscape of human mitochondrial lysine tRNA.

The folding energy landscape of RNA is greatly affected by interactions between the RNA and counterions that neutralize the backbone negative charges and may also participate in tertiary contacts. Valence, size, coordination number, and electron shell structure can all contribute to the energetic stabilization of specific RNA conformations. Using single-molecule fluorescence resonance energy transfer (smFRET), we have examined the folding properties of the RNA transcript of human mitochondrial tRNA(Lys), which possesses two different folded states in addition to the unfolded one under conditions of thermodynamic equilibrium.