Assembly of Escherichia coli50 S Ribosomes from Ribonucleic Acid and Protein Components

The in vitroassembly of Escherichia coli50 S ribosomes from the ribonucleic acid and protein components of 50 S ribosomes was attempted. The two components were prepared by subjecting 50 S ribosomes to LiCl-urea treatment. The ribonucleic acid fraction contains 23 S RNA and 5 S RNA in equimolar amounts and is free from significant amounts of protein. The protein fraction is composed of all of the protein subunits of 50 S ribosomes. The reconstitution experiments were carried out in a standard system: 0.01 mTris HCl, pH 7.5–0.3 mKCl-0.02 mmagnesium acetate-0.006 mβ-mercaptoethanol. The ribosomal RNA and protein components were incubated in the standard system at 42° for 15 min. These conditions permitted the formation of 30 S ribosomes from their components with 72% recovery of the original activity, as measured by polyuridylic acid-directed phenylalanine incorporation. Under identical conditions, the RNA and protein components of 50 S ribosomes were assembled stoichiometrically into 37 S particles. In a search for a chemical agent that would effectively reassemble particles of more compact structure, we found that spermine or spermidine or both, in addition to Mg++, led to the formation of 45 to 48 S ribonucleoprotein particles with all of the macromolecular components of 50 S ribosomes. The 45 to 48 S particles and 37 S particles were inactive in terms of polyuridylic acid-directed phenylalanine incorporation, the peptidyltransferase reaction and peptide chain termination reaction. We also showed that 23 S ribonucleoprotein particles are assembled in the absence of Mg++and polyamines. The particles continuously increase in sedimentation constant from 23 S to 26 S when Mg++is gradually increased to 10-2m. Above 10-2mMg++, 36 S particles appear and the sedimentation constant of the particles plateaus at 37 to 39 S at the concentration higher than 2 x10-2mMg++. Spermine or spermidine, in addition to Mg++, further increased the sedimentation constant of the particles continuously to 45 to 48 S. The process is the reversal of the previously reported unfolding of 50 S ribosomes by citrate-Mg++treatment. NH4Cl, in place of KCl, or the pH in a range between 7 and 8 had no significant effects on the ribosomal assembly. Mg++or Mg++plus polyamine(s) have to be added to the assembly system at zero time. The later those cations are added, the more unfolded are the resultant particles. The incorporation of 5 S RNA into the assembled ribonucleoprotein particles is stoichiometric and highly specific so that no significant incorporation of tRNA was observed.