Amino-acid incorporation into tRNA fragments and into heterologous combinations of fragments.
When the CCA-halves of tRNAAla1(yeast) and tRNAVal1(Escherichia coli) were incubated with the pG-halves of tRNAVal1 (E.coli) and trnaala1 (yeast), respectively, heterologous complexes were detected. When a 10-fold excess of one half was applied, up to 50% of the other half could be complexed. 5--12% alanine and valine incorporation was observed into the heterolgous combinations in which the pG-halves were derived from tRNAAla1 (yeast) and tRNAVal1 (E.coli), respectively. Although the values are small they appear to be significant considering the results of a number of control experiments. The CCA-half of tRNASer1,2(yeast) and another fragment of this tRNA which extends from the dihydrouridine region to the CCA-terminus were inactive in the aminoacylation assay but they could be converted into a form which accepted serine under standard conditions even in the absence of a complementary fragment. One activation procedure involved the addition of MgCl2 to Mg2+-free fragment solutions, the other consisted in a brief heating-cooling cycle of the fragment solutions at low Mg2+ concentrations. With the two procedures up to 20% or up to 40%, respectively, of the maximal serine incorporation were achieved. At 37 degrees C the active conformation of the fragments persisted only for a few minutes. Analogously, the CCA-halves of tRNAPhe (yeast), tRNAAla1 (yeast), and tRNAVal1 (E.coli)could be activated although here the extent of aminoacylation varied greatly from one experiment to the other. Mischarging of the activated CCA-halves of tRNASer1,2 (yeast) and tRNAPhe (yeast) with phenylalanine and serine, respectively, was not observed. The results obtained with the hererologous fragment combinations and with the CCA-halves alone, which at first sight seem to contradict each other, are discussed with respect to the conformational requirements of synthetase-tRNA recognition.
D000409:Alanine; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010649:Phenylalanine; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012694:Serine; D012714:Serine-tRNA Ligase / Q000378:metabolism; D013045:Species Specificity; D013696:Temperature; D046249:Transfer RNA Aminoacylation / Q000187:drug effects; D014633:Valine
D012694:Serine; D010649:Phenylalanine; D012343:RNA, Transfer; D012714:Serine-tRNA Ligase; D010652:Phenylalanine-tRNA Ligase; D014633:Valine; D008274:Magnesium; D000409:Alanine