Higher-order structure of bovine mitochondrial tRNAPhe lacking the ‘conserved’ GG and TΨCG sequences as inferred by enzymatic and chemical probing

Bovine mitochondrial (mt) phenylalanine tRNA (tRNAPhe), which lacks the ‘conserved’ GG and TΨYCG sequences, was efficiently purified by the selective hybridization method using a solid phase DNA probe. The entire nucleotide sequence of the tRNA, including modified nucleotides, was determined and its higher-order structure was investigated using RNaseT2 and chemical reagents as structural probes. The D and T loop regions as well as the anticodon loop region were accessible to RNaseT2, and the N-3 positions of cytidines present in the D and T loops were easily modified under the native conditions in the presence of 10mM Mg2+. On the other hand, the nucleotides present in the extra loop were protected from the chemical modification under the native conditions. From the results of these probing analyses and a comparison of the sequences of mitochondrial tRNAPhe genes from various organisms, it was inferred that bovine mt tRNAPhe lacks the D loop/T loop tertiary interactions, but does have the canonical extra loop/D stem interactions, which seem to be the main factor for bovine mt tRNAPhe to preserve its L-shaped higher-order structure.