Quantification of the different ribosomal phases during the translational elongation cycle in rabbit reticulocyte lysates.

The proportionality of different ribosomal phases during elongation was estimated in the highly effective rabbit reticulocyte lysate system by use of several complementary analytical methods. The stoichiometric amounts of ribosome-bound elongation factors EF-1 and EF-2 were determined as a measure of ribosomal A-site occupation. The results were correlated with the puromycin reactivity of the P-site-located nascent polypeptide chains. Approximately 25% of the ribosomes were associated with EF-2, indicating that their A-sites contained peptidyl-tRNA. About 35% were associated with EF-1, signifying that their A-sites were occupied by aminoacyl-tRNA. The puromycin reactivity of the nascent chains was approximately 40%. From these data it is concluded that 75% of the peptidyl-tRNAs were located in the P-sites and that their puromycin reactivity was limited by the availability of ribosomal A-sites free for puromycin interaction. After guanosine 5'-[beta, gamma-methylene]triphosphate blockage of the translation, the ribosomal content of elongation factors drastically changed. Under these conditions the proportion of EF-1-containing ribosomes increased to approximately 50% while EF-2-containing ribosomes decreased to 5%. Concomitantly, the puromycin reactivity increased to approximately 45%. In contrast to previous assumptions the experiments support the view that the elongation rate is limited by the availability of ribosomal A-sites for the selection of mRNA-cognate aminoacyl-tRNAs.