The third ribosomal tRNA-binding site, the E site, is occupied in native polysomes |
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Authors: | J Remme T Margus R Villems K H Nierhaus |
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Institution: | Institute of Chemical Physics and Biophysics, Academy of Sciences, Estonian SSR, Tartu. |
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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 a buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37 degrees 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. |
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