Inhibitors of the Transfer of Amino Acids from Aminoacyl Soluble Ribonucleic Acid to Proteins

J.M Clark,Albert Yen Chang

doi:10.1016/s0021-9258(18)97017-1

J.M Clark, Albert Yen Chang

Open Access

https://doi.org/10.1016/s0021-9258(18)97017-1

Copy DOI

Abstract

SUMMARY Puromycin, gougerotin, neomycin B, tetracycline, chloram- phenicol, and cycloheximide inhibit protein synthesis in various cell-free systems. Assays designed to determine the mechanism of action of these inhibitors indicat.e (a) that tetracycline inhibits the binding of aminoacyl soluble ribonucleic acid to messenger ribonucleic acid-containing ribosomes; (b) that the aminoacyl nucleoside, puromycin, releases peptide material from peptidyl soluble ribonucleic acid, messenger ribonucleic acid, ribosome complexes while the related aminoacyl nucleoside, gougerotin, blocks this release; (c) that neomycin B alters the binding of aminoacyl soluble ribonucleic acid onto messenger ribonucleic acid, ribosome complexes; (d) that chloramphenicol can compete with a messenger ribonucleic acid for sites on bacterial ribosomes; and (e) that cycloheximide does not influence the binding of aminoacyl soluble ribonucleic acid to ribosomes or the puromycin- dependent release of peptides from pept’idyl soluble ribonucleic acid, messenger ribonucleic acid, ribosome complexes. Aclcnowledgments-We wish to thank Dr. Richard Schweet for his encouragement of this work and Mrs. Ronnie Barker for invaluable technical assistance. REFERENCES

Highlights

This paper presents a general picture of the sites of action of various inhibitors of the transfer of amino acids from aminoacyl S-RNA t,o protein
Tables I and II emphasize that puromycin, gougerotin, tetracycline, and neomycin are transfer react,ion inhibitors in both mammalian and bacterial systems while cycloheximide and chloramphenicol fail to inhibit the transfer reactions in E. coli and rabbit reticulocyte systems, respectively
This interference appears to be a tetracycline-dependent inhibition of the association of aminoacyl S-RNA with messenger RNA on ribosomes for (a) tetracycline does not compete with messenger RNA for sites upon ribosomes (Fig. 1) (17, 22); (b) puromycin peptide formation, which does not appear to require additional binding of aminoacyl S-RNA into ribosome complexes, is not markedly influenced by tetracycline (Table IV); and (c) the completion of partial chains, which does require additional aminoacyl S-RNA binding into ribosome complexes, is inhibited by t,etracycline (Table IV)

Summary

PROCEDURE

Phenylalanyl S-RNA’ or “C-~-lysyl S-RNA were prepared with the use of the E. coli 100,000 x g supernatant of Nirenberg, Matthaie, and Jones (14) and E. coli S-RNA (General Biochemical Industries) under the conditions of Holley et al (15). The resultant 14C-L-phenylalanyl S-RNA or 14C-lysyl S-RNA was isolated by phenol extraction (specific activity, 3.6 to 4.4 mpmoles of 1%.L-phenylalanine and 5.2 mpmoles of 14C-L-lysine per mg). S-RNA with the cell-free system of Nirenberg et al (14) with the following modification. Clark and Gunther (5) except in cases of polylysine synthesis, in which 5% trichloroacetic acid, pH 1.7, containing 0.025%. New Zealand rabbits according to the method of Allen and Schweet (16). Inhibition of hemoglobin synthesis and poly r-dependent synthesis of polyphenylalanine in cell-free rabbit reticulocyte systems was studied by t.he assay procedure of Allen and Schweet (16) with the modification that in cases of 14C-. Polyphenylalanine synthesis, 50 pmoles of 14C-L-phenylalanine (1 X lo[5] cpm per pmole), 50 pg of poly U, and ribosomes washed

The abbreviations used are

Procedure”

Findings

DISCUSSION

SUMMARY