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

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Abstract
Bovine mitochondrial (mt) phenylalanine tRNA (tRNA(Phe)), which lacks the 'conserved' GG and T Psi YCG 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 it higher-order structure was investigated using RNase T-2 and chemical reagents as structural probes. The D and T loop regions as well as the anticodon loop region were accessible to RNase T-2, 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 form 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.