The Protection by 70 S Ribosomes of N‐Acyl‐aminoacyl‐tRNA against Cleavage by Peptidyl‐tRNA Hydrolase and its Use to Assay Ribosomal Association

Abstract

Free N‐carbobenzyloxy‐phenylalanyl‐tRNA is rapidly cleaved by the enzyme peptidyl‐tRNA hydrolase. When bound to a 30 S ribosome in the presence of poly(U), the substrate is hydrolyzed as rapidly as when free. The addition of 50 S ribosomal subunits to form the 70 S ribosomal binding complex protects the bound substrate from the enzyme. The degree of protection depends on the state of activity of the 50 S subunit. Under conditions favoring the formation of 70 S ribosomes, active 50 S subunits (capable of catalyzing the peptidyl transferase reaction) afford complete protection at one mole per mole of 30 S subunits. Inactive 50 S subunits also protect the substrate, but less effectively. The protection of bound substrate can be used to measure the amount of 70 S ribosomal binding complex formed and the rate of its formation. Using highly active hydrolase preparations, we have developed a rapid assay procedure and present several examples of its use. Under conditions favoring the association of ribosomal subunits, the formation of the 70 S complex may reach completion within a few seconds. Copyright © 1971, Wiley Blackwell. All rights reserved