title: Quantification of the different ribosomal phases during the translational elongation cycle in rabbit reticulocyte lysates.
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 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.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002474:Cell-Free System; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism*; D011691:Puromycin; D012343:RNA, Transfer / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000502:physiology*; D012270:Ribosomes / Q000378:metabolism
chemical_list: D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D011691:Puromycin; D012343:RNA, Transfer