Peptide Chain Elongation: Indications for the Binding of an Amino Acid Polymerization Factor, Guanosine 5′-Triphosphate–Aminoacyl Transfer Ribonucleic Acid Complex to the Messenger–Ribosome Complex

The intermediate steps in peptide chain elongation were studied in a cell-free system in which polyuridylic acid directs the formation of polyphenylalanyl transfer ribonucleic acid from phenylalanyl transfer ribonucleic acid. The system consists of guanosine 5′-triphosphate, salts, Escherichia coli ribosomes, and three amino acid polymerization factors (S1, S2, and S3) from Bacillus stearothermophilus. (The factors from B. stearothermophilus correspond to the following factors from E. coli: Si to Ts, S2 to G, and S3 to Tu.) The incubation of S1, S3, guanosine 5′-triphosphate, and phenylalanyl transfer ribonucleic acid results in the formation of a S3–guanosine 5′-triphosphate–phenylalanyl transfer ribonucleic acid complex (complex II). The incubation of complex II with a ribosome–polyuridylic acid–peptidyl transfer ribonucleic acid complex (actually acetylphenylalanyl transfer ribonucleic acid was used instead of peptidyl transfer ribonucleic acid) leads to the cleavage of guanosine 5′-triphosphate and the formation of an S3–guanosine 5′-diphosphate complex and inorganic phosphate, as well as the synthesis of acetyldiphenylalanyl transfer ribonucleic acid. 5′-Guanylylmethylenediphosphonate, an analog of guanosine 5′-triphosphate which cannot be cleaved to guanosine 5′-diphosphate and inorganic phosphate, was substituted for guanosine 5′-triphosphate in an incubation mixture also containing S1, S3, phenylalanyl transfer ribonucleic acid, and the ribosome–polyuridylic acid–acetylphenylalanyl transfer ribonucleic acid complex. Under these conditions, approximately equimolar amounts of S3, 5′-guanylylmethylenediphosphonate, and phenylalanyl transfer ribonucleic acid became bound and remained attached to ribosomes, and acetyldiphenylalanyl transfer ribonucleic acid was not formed. These results may reflect the transient existence of a ribosome-bound S3–guanosine 5′-triphosphate–phenylalanyl transfer ribonucleic acid complex as an intermediate preceding acetyldiphenylalanyl transfer ribonucleic acid synthesis. The short life span of this hypothetical intermediate may be due to the rapid cleavage of the guanosine 5′-triphosphate molecule in it, which results in the release of an S3–guanosine 5′-diphosphate complex and inorganic phosphate from the ribosome and acetyldiphenylalanyl–transfer ribonucleic acid formation.