Aminoacylation of Fragment Combinations from Yeast tRNAPhe.

The aminoacylation of 15 fragments from yeast tRNA^Phe and of various mixtures of these fragments was studied in detail. The aminoacylation conditions were systematically varied in order to obtain maximal incorporation of phenylalanine. No fragment by itself accepted phenylalanine. Fragment combinations in which parts of the anticodon loop or the dihydrouridine loop were missing, accepted phenylalanine to an extent of 20–75% compared to intact tRNA^Phe. Modifications in both regions together inactivated the molecule. With the anticodon stem and/or the manilooop missing, low but significant activity was found. Splits in the dihydrouridine loop and in the T-ψ-C loop together did not abolish the phenylalanine acceptance. Combinations in which parts of the dihydrouridine stem or of the T-ψ-C stem were missing could not be aminoacylated. Complete association of complementary fragments was observed in all cases. The results of charging and heating experiments, however, indicate the coexistence of different flagment combinations in a fragment mixture. The incomplete aminoaeylation of fragment mixtures is explained by the presence of fragment combinations with an unsuitable three-dimensional structure. The rate of aminoacylation was lower for fragment combinations than for tRNA^Phe. The Michaelis constants of 3 fragment combinations were identical to the one of t RNAPne. Two fragment mixtures inhibited the rate of charging of tRNA^Phe. Phenylalanine was the only amino acid which was accepted by the fragment mixtures with synthetases from yeast or Escherichia coli. No miseharging was observed. The results together with those of related publications are discussed with respect to recognition sites of tRNA^Phe for the cognate synthetase. [ABSTRACT FROM AUTHOR]