Three tRNA binding sites on Escherichia coli ribosomes.

Publisher Website
Google Scholar
Abstract
The binding of N-acetyl-Phe-tRNAPhe (an analog of peptidyl-tRNA), Phe-tRNAPhe and deacylated tRNAPhe to poly(U)-programmed tightly coupled [E. coli] 70S ribosomes was studied. The N-acetyl-Phe-tRNAPhe binding is governed by an exclusion principle: not more than 1 N-acetyl-Phe-tRNAPhe can be bound per ribosome, although this peptidyl-tRNA analog can be present either at the aminoacyl-tRNA (A) site or the peptidyl-tRNA (P) site. Two Phe-tRNAPhe molecules are accepted by 1 ribosome in the presence of poly(U). This aminoacyl-tRNA binds enzymatically (in the presence of elongation factor Tu and GTP) and nonenzymatically to the A site and is then transferred to the P site, if that site is free. If this elongation factor G-independent movement is hampered, either by using an incubation temperature of 0.degree. C or by the addition of the translocation inhibitor viomycin, only 1 Phe-tRNAPhe per ribosome can be bound. The effect of the peptidyltransferase inhibitor chloramphenicol on the binding is similar to that of viomycin. In the absence of poly(U), Phe-tRNAPhe cannot bind to the ribosome. Deacylated [14C]tRNAPhe can bind in 3 copies to 1 ribosome. The new 3rd tRNA binding site is called the E site. The sequence of filling the sites is P, E and A. The apparent binding constants for the P and the E sites are both .apprx. 9 .times. 106 M-1 and that for the A site is 1.3 .times. 106 M-1. In the absence of poly(U), only 1 deacylated tRNAPhe can be bound per ribosome. This tRNAPhe most likely occupies the P site.