Protein synthesis by hybrid ribosomes reconstructed from rabbit reticulocyte ribosomal core-particles and amphibian or fungal split-proteins.
It was shown that high-salt (2.75 M-NH4Cl/69mM-MgCl2) shock treatment at 0 degrees C of the larger subparticles (L-subparticles) of rabbit, Xenopus laevis and Neurospora crassa cytoplasmic ribosomes yielded split-protein fractions that were not only functionally equivalent but also interchangeable. Thus, although the remaining core-particles were inactive in both the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis, activity was restored when they were combined with either homologous or heterologous split-protein fractions. This technique was used to prepare active hybrid L-subparticles, e.g. rabbit cores/N. crassa split-proteins, and also active hybrid ribosomes, e.g. rabbit smaller subparticle/X. laevis core-particle/rabbit split-proteins. Rabbit and X. laevis split-protein fractions labelled with 14C by reductive methylation with [14C]formaldehyde and sodium cyanoborohydride were both shown to bind to rabbit core-particles in approximate correlation with the degree of re-activation. The split-protein fractions of rabbit and X. laevis L-subparticles were analysed by two-dimensional and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights (measured in sodium dodecyl sulphate gels) of the split-proteins of rabbit and X. laevis L-subparticles were found to be similar. These results demonstrate that the peptidyltransferase active centre of cytoplasmic ribosomes of eukaryotes has components that are interchangeable over a wide evolutionary range. Evidently the essential molecular architecture of the active centre is highly conserved.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008970:Molecular Weight; D009492:Neurospora crassa; D010452:Peptide Biosynthesis; D010455:Peptides; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000096:biosynthesis; D011691:Puromycin; D011817:Rabbits; D012156:Reticulocytes / Q000648:ultrastructure; D012269:Ribosomal Proteins / Q000096:biosynthesis*; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure; D014981:Xenopus
D010455:Peptides; D012269:Ribosomal Proteins; C012001:polyphenylalanine; D010649:Phenylalanine; D011691:Puromycin