The third ribosomal tRNA‐binding site, the E site, is occupied in native polysomes

Abstract

The nucleic acids of Escherichia coli cells were uniformly labelled with 32P by growing the cells in [32P]orthophosphoric acid for about four generations. The cells were harvested in the logarithmic phase, resuspended in á buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37°C in the presence of 3H‐labelled amino acids. This procedure preferentially labels growing peptidyl chains. Polysomes were isolated, the fraction in the post‐translocational state was assessed by a puromycin reaction and the tRNA content/70S ribosome was quantified in comparison to the amount of 5S rRNA determined after separation by gel electrophoresis. The data revealed that at least 75% of post‐translocational ribosomes in isolated native polysomes carry a tRNA in their E site. The results are consistent with the allosteric three‐site model for the elongation cycle but disagree with the two‐site model. Copyright © 1989, Wiley Blackwell. All rights reserved