Higher-order structure of bovine mitochondrial tRNA^Phe lacking the 'conserved' GG and TψCG sequences as inferred by enzymatic and chemical probing

Bovine mitochondrial (mt) phenylalanine tRNA (tRNA^Phe), 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 tRNA^Phe genes from various organisms, it was inferred that bovine mt tRNA^Phe 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 tRNA^Phe to preserve its L-shaped higher-order structure.