The Inhibition Pattern of Antibiotics on the Extent and Accuracy of tRNA Binding to the Ribosome, and Their Effect on the Subsequent Steps in Chain Elongation

Abstract

Ac[14C]Phe‐tRNA (cognate) and Ac[3H]Leu‐tRNA (miscognate) were bound as competing peptidyl‐tRNA analogues to tightly coupled ribosomes programmed with poly(U). The test system allows the determination of the accuracy of tRNA selection at the A and P site, as well as the measurement of the peptidyltransferase and translocation reactions in the presence of various antibiotics. The following results were obtained. Cognate tRNA could be bound to either the A or P site with nearly 10% specificity. In contrast, miscognate tRNA could be exclusively bound to the P site but poorly to the A site. The accuracy of tRNA selection (extent of binding) was sixfold higher at the A site than at the P site. The extent of A site binding for both cognate and miscognate tRNA was severely inhibited by viomycin, tetracycline and neomycin, and to a lesser extent by streptomycin. No inhibition of binding at the P site was found with all antibiotics tested. This finding, and that of the preceding point, is taken as evidence that acetylated aminoacyl‐tRNA (an analogue for a peptidyl‐tRNA) has direct access to the P site and does not reach this site via a transient A site binding. In addition to the known effects of streptomycin and neomycin on tRNA selection at the A site, viomycin was found to enhance drastically the error fraction at this site. The peptidyltransferase is inhibited by the drugs in the following order: chloramphenicol > viomycin, neomycin > streptomycin. Equivalent results were found with cognate and miscognate tRNA. The translocation reaction was severely inhibited by viomycin and neomycin and to a lesser extent by streptomycin. A striking correspondence was found between the error‐inducing capacity (distortion of codon‐anticodon interaction at the A site) and the inhibition of the translocation reaction by various drugs. This finding suggests that codon‐anticodon interaction at the ribosomal A site is an important if not essential prerequisite for the translocation reaction. Copyright © 1983, Wiley Blackwell. All rights reserved