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

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Abstract
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 .apprx. 40%. The data indicate that 75% of the peptidyl-tRNA 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 .apprx. 50%, while EF-2-containing ribosomes decreased to 5%. Concomitantly, the puromycin reactivity increased to .apprx. 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-tRNA.