A new technique was developed for measuring the amount of peptidyl-tRNA in a protein-synthesizing system in vitro. By this technique the course of the puromycin reaction may be followed and the modes of action of various inhibitors of protein synthesis readily determined. We conclude that the polypeptide alpha sarcin inhibits the binding of aminoacyl-tRNA into the ribosomal ‘A’ site, that sparsomycin inhibits the peptidyl transferase reaction and that cycloheximide may block translocation.

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