title: Partial reassembly of yeast 60 S ribosomal subunits in vitro following controlled dissociation under nondenaturing conditions.
abstract: Previously it has been shown that 12 of the yeast ribosomal proteins were extractable from 60 S subunits under a specific nondenaturing condition [J. C. Lee, R. Anderson, Y. C. Yeh, and P. Horowitz (1985) Arch. Biochem. Biophys. 237, 292-299]. In the present paper, we showed that these proteins could be reassembled with the corresponding protein-deficient core particles to form biologically active ribosomal subunits. Effects of time, temperature, and varying concentrations of monovalent cations, divalent cations, cores, and ribosomal proteins on reconstitution were examined. Reconstitution was determined by binding of radiolabeled proteins to the nonradiolabeled cores as well as activity for polypeptide synthesis in a cell-free protein-synthesizing system. The optimal conditions for reconstitution were established. Whereas the core particles were about 10-20% as active as native 60 S subunits in an in vitro yeast cell-free protein-synthesizing system, the reconstituted particles were 80% as active. The activity of the reconstituted particles was proportional to the amount of extracted proteins added to the reconstitution mixture. About 55 +/- 7% of the core particles recombined with the extracted proteins to form reconstituted particles. These reconstituted particles cosedimented with native 60 S subunits in glycerol gradients and contained all of the 12 extractable proteins.
mesh_terms: D000643:Ammonium Chloride; D002499:Centrifugation, Density Gradient; D005656:Fungal Proteins / Q000302:isolation & purification*; D007460:Iodoacetamide; D011489:Protein Denaturation; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D012269:Ribosomal Proteins; D000643:Ammonium Chloride; D007460:Iodoacetamide