Soluble Amino Acid-incorporating System

III. FURTHER STUDIES ON THE PRODUCT AND ITS RELATION TO THE RIBOSOMAL SYSTEM FOR INCORPORATION

From the ¹ From the Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

The soluble amino acid incorporation system from Escherichia coli was fractionated by density gradient centrifugation and was shown to contain aminoacyl soluble ribonucleic acid (S-RNA) transfer factor, aminoacyl S-RNA synthetase, and acceptor molecules which accept amino acids from aminoacyl S-RNA, but direct evidence that aminoacyl S-RNA transfer factor participates in the soluble system is still lacking. The acceptor molecules are shown to be present also on the ribosomes. Thus the ¹⁴C-leucine or ¹⁴C-phenylalanine-labeled product of the soluble system was identical with the major product of the ribosomal system obtained in the absence of added messenger RNA. The product of the soluble amino acid incorporation system was digested by trypsin and subjected to paper chromatography and paper electrophoresis. At least four different radioactive components were obtained by these procedures.

When ¹⁴C-leucine and ¹⁴C-phenylalanine are incorporated by the ribosomal system, without addition of messenger RNA, the major portion of the incorporated amino acids had free amino groups which were revealed by the dinitrofluoro-benzene technique for NH₂-terminal analysis. This observation suggests that even with the conventional ribosomal system, if the incorporation was studied in the absence of added messenger, the major portion of ¹⁴C-leucine and ¹⁴C-phenylalanine incorporation is an addition of amino acid to the NH₂ group of a preformed acceptor or to a lysine NH₂ group in the internal linkage. This incorporation is tentatively called "NH₂-group addition" to distinguish it from the usual incorporation representing a chain elongation through successive addition of amino acid at the COOH-terminal end. The NH₂ group addition involving ribosomes is dependent on aminoacyl S-RNA, soluble enzymes, and the acceptor substance on ribosomes. It was sensitive to RNase and puromycin but was not dependent on added guanosine triphosphate or messenger RNA. Some release of radioactive product takes place from the ribosomes during the incorporation reaction.

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