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.
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