Aminoacylation of fragment combinations from yeast tRNA phe .

Abstract

The aminoacylation of 15 fragments from yeast tRNAPhe 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 tRNAphe. Modifications in both regions together inactivated the molecule. With the anticodon stem and/or the minilooop 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 amino‐acylated. Complete association of complementary fragments was observed in all cases. The results of charging and heating experiments, however, indicate the coexistence of different fragment combinations in a fragment mixture. The incomplete aminoacylation 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 tRNAPhe. The Michaelis constants of 3 fragment combinations were identical to the one of tRNAPhe. Two fragment mixtures inhibited the rate of charging of tRNAPhe. Phenylalanine was the only amino acid which was accepted by the fragment mixtures with synthetases from yeast or Escherichia coli. No mischarging was observed. The results together with those of related publications are discussed with respect to recognition sites of tRNAPhe for the cognate synthetase.