Reassembly of the peptidyltransferase centre of larger subparticles of rabbit reticulocyte ribosomes from a core-particle and split-protein fraction

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Abstract
The reconstruction, from a core-particle and split-protein fraction, of the larger subribosomal particle of rabbit reticulocytes is reported. The reassembled particle was active in polyphenylalanine synthesis and in the puromycin reaction. The core-particles and split-protein fractions were obtained by treatment of the larger subparticle with salt solutions containing NH4+ and Mg2+ in the molar ratio 40:1 over the range 2.25-2.75 M-NH4Cl/56-69 mM-MgCl2 at 0.degree. C. This treatment led to the loss of about 8 proteins (approximately 17% of the protein moiety), which were found wholly or largely in the split-protein fraction as shown by 2-dimensional gel electrophoresis. The core particle retained 5S rRNA and had much decreased (no more than 10% of control) ability to function in the puromycin reaction or in poly(U)-directed polyphenylalanine synthesis. Activity was recovered when the recombined core-particle and split-protein fractions were dialysed overnight at 4.degree. C against 0.3 M-NH4Cl/15 mM-MgCl2/1 mM-dithiothreitol/15% (vol/vol) glycerol/20 mM-Tris/HCl, pH 7.6 and then heated for 1 h at 37.degree. C. The recovery was 40-80% of the original activity. Raising the concentration of MgCl2 to 300 mM in 2.5 M-NH4Cl led to the removal of 7 rather than 8 proteins, and the core particle remained active in the puromycin reaction. The protein retained by raising the concentration of Mg2+ is probably an essential component of the peptidyltransferase center of the ribosome.