Protein synthesis by hybrid ribosomes reconstructed from rabbit reticulocyte ribosomal core-particles and amphibian or fungal split-proteins

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Abstract
High-salt (2.75 M NH4Cl/69 mM MgCl2) shock treatment at 0.degree. 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 labeled with 14C by reductive methylation with [14C]formaldehyde and sodium cyanoborohydride both bound 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 analyzed by 2-dimensional and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The MW (measured in sodium dodecyl sulfate gels) of the split-proteins of rabbit and X. laevis L-subparticles were similar. The peptidyltransferase active center of cytoplasmic ribosomes of eukaryotes apparently has components that are interchangeable over a wide evolutionary range. Evidently the essential molecular architecture of the active center is highly conserved.