Species-specific Reactions of Cell-free Polypeptide Synthesis

Abstract

Summary Species-specific steps of polypeptide biosynthesis were studied in cell-free systems with components from E. coli , rat liver, and etiolated pea seedlings. For aminoacyl-tRNA synthesis, enzyme and tRNA fractions from Euglena gracilis and Anacystis nidulans were also included. Both amino acid acceptor and transfer reactions appeared specific in respect to the protokarytic or eukaryotic origins of the macromolecular components. According to the first reaction, the degree of specificity between protokaryotes ( Anacystis ) and eukaryotes ( Euglena , rat liver) was further dependent on the amino acid species. With aminoacyl-tRNA synthetases from the blue-green alga and tRNAs from rat liver and green as well as u. v.-bleached Euglena cells, the rates of arginyl-tRNA formation were at least in the same order of the homologous system from Anacystis. In contrast, a strict class specificity existed for phenylalanine and leucine acceptor activities. Transfer RNA from Anacystis was not charged with arginine by the eukaryotic synthetases, and the Euglena enzymes specific for leucine-tRNA showed a complete homologous behaviour to the tRNAs from any source. Poly U-dependent 14 C-phenylalanine incorporation with the homologous pea seedling system was found inactive, however, the heterologous system consisting of pea ribosomes and rat liver cytoplasm did work. On the other hand, the poly U-directed 14 C-phe-tRNA transfer into polypeptides occurred in all homologous, and in the heterologous systems from eukaryotes. Since the rates of 14 C-phe-tRNA transfer into polypeptides were additive also in combination with rat liver and pea seedling supernatants, it was concluded that the block of synthesis caused by pea supernatant fractions must be localized prior to the transfer step. Transfer activity was absent in heterologous systems with E. coli components. Additional rat liver supernatant fraction to the homologous E. coli system did not disturb the transfer reaction, but E. coli supernatants caused a marked suppression of the activity in the homologous eukaryotic system.