Protein synthesis in a cell-free system prepared from human placenta II. pH 5 enzyme inefficiency due to defects in tRNA charging with resulting loss of elongation factor 1

Abstract

Washed ribosomes and the pH 5 fraction of cell sap were isolated from human placenta and from rat liver and used for preparing a system for in vitro amino acid incorporation. In such a system, ribosomes prepared from human placenta and from rat liver showed the same capacity for amino acid incorporation, but the placental pH 5 fraction was one-third as effective as the pH 5 fraction of rat liver. In order to identify limiting factors in the placental pH 5 enzyme, its efficiency in attaching amino acids to tRNA and subsequently transferring amino acids from aminoacyl-tRNA to ribosomes was examined. Crude preparations of aminoacyl-tRNA synthetases were made from placenta and from rat liver and were compared for their capacity to charge rat liver tRNA with amino acids. The placental synthetase preparation was inferior in charging activity. In addition, tRNA isolated from full-term placenta had a reduced capacity for accepting amino acids in the presence of rat liver synthetase and especially in the presence of the placental synthetase preparation. Thus defective charging of tRNA contributes to the inefficiency of the placental pH 5 fraction in cell-free protein synthesis. The pH 5 fractions of human placenta and of rat liver were then compared for their ability to promote peptide chain elongation by transfer of amino acids from pre-formed aminoacyl-tRNA to nascent protein chains on rat liver ribosomes. The placental pH 5 fraction was much less effective in carrying out transfer, but could be fully corrected by adding elongation factor 1 to the preparation. The deficiency of elongation factor 1 in the placental pH 5 fraction was confirmed by comparing the ability of the pH 5 enzyme from placenta and from rat liver to stabilize aminoacyl-tRNA against spontaneous hydrolysis in vitro. Since only part of the elongation factor 1 of cell sap precipitates at pH 5, the amount of elongation factor 1 remaining in the supernatant fraction was assayed with stripped ribosomes. The supernatant fraction left after precipitation of the placental cell sap at pH 5 was found to have 70% of the elongation factor 1 content of the same fraction prepared from rat liver. Since the transfer capacity of the pH 5 enzyme fraction made from the placental cell sap was much more severely reduced, this suggested failure of elongation factor 1 to precipitate at pH 5 from placental cell sap. This was confirmed and shown to be due to the lack of charged tRNA in placental cell sap noted above. When rat liver tRNA was added to the placental cell sap before adjusting to pH 5, the precipitate obtained at pH 5 now contained more elongation factor 1, while the supernatant fraction had less elongation factor 1 activity. This effect was found to be specific for charged tRNA. A scheme is proposed whereby the amounts of certain cell sap factors may be coordinated in vivo in relation to the charging of tRNA.