The third ribosomal tRNA-binding site, the E site, is occupied in native polysomes.
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.
Remme
J
J
Institute of Chemical Physics and Biophysics, Academy of Sciences, Estonian SSR, Tartu.
Margus
T
T
Villems
R
R
Nierhaus
K H
KH
D001665:Binding Sites; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011132:Polyribosomes / Q000378:metabolism*; D011691:Puromycin / Q000494:pharmacology; D012341:RNA, Ribosomal, 5S / Q000302:isolation & purification / Q000378:metabolism; D012343:RNA, Transfer / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D010761:Phosphorus Radioisotopes; D012341:RNA, Ribosomal, 5S; D011691:Puromycin; D012343:RNA, Transfer