A Counterintuitive Mg²⁺-dependent and Modification-assisted Functional Folding of Mitochondrial tRNAs

Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg²⁺ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg²⁺ is critical for folding of the truncated D-domain of bovine mtRNA^Met with the tRNA's T-domain. Contrary to the expectation that Mg²⁺ stabilizes RNA folding, the mtRNA^Met D-domain structure was unfolded and relaxed, rather than stabilized in the presence of Mg²⁺. Because the D-domain is transcribed prior to the T-domain, we conclude that Mg²⁺ prevents misfolding of the 5′-half of bovine mtRNA^Met facilitating its correct interaction with the T-domain. The interaction of the mtRNA^Met D-domain with the T-domain was enhanced by a pseudouridine located in either the D or T-domains compared to that of the unmodified RNAs (K_d = 25.3, 24.6 and 44.4 μM, respectively). Mg²⁺ also affected the folding interaction of a yeast mtRNA^Leu1, but had minimal effect on the folding of an Escherichia coli cytoplasmic tRNA^Leu. The D-domain modification, dihydrouridine, facilitated mtRNA^Leu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.