Abstract
Bovine mitochondrial (mt) phenylalanine tRNA (tRNAPhe) was purified on a large scale using a new hybridization assay method developed by the authors. Although its melting profile suggested a loose higher order structure, presumably influenced by the apparent loss of D loop-T loop interaction necessary for forming a rigid L-shaped tertiary structure, its aminoacylation capacity catalyzed by mt phenylalanyl-tRNA synthetase (PheRS) was nearly equal to that of Escherichia coli tRNAPhe. Misaminoacylation was not observed for the mt tRNAPhe-mt PheRS system. Comparing the aminoacylation efficiencies of several combinations of tRNAPheS and PheRSs from various sources, including bovine mitochondria, bovine and yeast cytosols, E. coli, Thermus thermophilus, and Sulfolobus acidocaldarius, it was clarified that mt PheRS was able to aminoacylate all the above mentioned tRNAPhe species, albeit with varying degrees of efficiency. This broad charging spectrum suggests that mt PheRS possesses a relatively simple recognition mechanism toward its substrate, tRNAPhe.
Highlights
Bovinme itochondria(lmtp)henylalaninteRNA tRNAsona scale large enough for biochemical studiesin as purified on a large scale using a new order to elucidate the functional significance of their unusual hybridization assay method developed by the authors. structures
Acidocaldarius, it was clarified that mt PheRS was aminoacylate structurally unusual mt tRNAPh“but able to aminoacylateall the above mentioned tRNAPhealso bacterial and cyt tRNAPh%from various sources
For estimation of the K, values, each tRNA concentration was resulted ina 2-3 times increase of its charging activity, calculated on the basis of the following molar extinction coefficients: 7200 and 6600 for E. coli and yeast tRNAPhe,respectively [51]; that for mt tRNAPhewasapproximated to be the same as that for mt tRNA&'ACy (8600, Ref. 19) on the basis of the observation that the melting profiles of these two tRNAs resemble each other; that for S. acidocaldarius tRNAPhewas approximated to be the same whereas no change was observed for the PheII and -111 fractions
Summary
Various animals has revealed that animal mt DNA encodes Specificity of Synthetic DNA Probes in Hybridization Assimple translation machineries, which depend partly on cy- say-Two synthetic DNA probes complementary tothe tosolic (cyt) translation systems [1,2,3,4,5,6,7,8,9]. Each view of the absence of invariant nucleotides such as GG in probe hybridized only to the corresponding unfractionated the D loop and T+CG/AA in the T loop, necessary for tertiary tRNA (tRNA””, see Fig. 5 in Miniprint), and thehybridized interaction between both these loops [10]. As an extremely spot was detected even when each tRNA”’ was diluted by unusual example, mammalian mt serine tRNAs,which specify times. This indicates cross-contaminationto be virtually the AGY codon (tRNA&), are known to lack all parts of the negligible.
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