Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins.

Ribosomes are structurally complex cell organelles consisting of ribosomal RNA and a number of different protein molecules. The complexity of these particles raises two major questions: What is the relationship between structure and function of the ribosome, and what is the mechanism of assembly of the ribosome? It is obvious that the study of these two questions would be greatly aided if a method could be found for assembling active ribosomes from their dissociated macromolecular constituents. Partial dissociation of ribosomes into inactive smaller ribonucleoprotein particles ("core" particles) and proteins ("split proteins") and the reconstitution of functionally active ribosomal particles from these components were reported previously.'-3 This system has been used in this laboratory to study the two problems mentioned above. Split proteins were fractionated and various artificial ribosome derivatives deficient in specific protein components were prepared.4-6 Analysis of the activity of these particles has revealed the functional significance of each of the purified ribosomal split proteins. Some proteins have been shown to be essential for ribosomal functions, others to be dispensable but to have stimulatory activity in in vitro polypeptide synthesis. It has also been shown that assembly of ribosomes from the split proteins and the core particles is spontaneous, supporting the hypothesis of spontaneous selfassembly of ribosomes from subribosomal components.4' 7 In extending this type of approach further, we have now been able to dissociate 23S core particles (derived from 30S ribosomal particles) into free ribosomal RNA (16S RNA) and proteins ("core proteins" from 30S, designated as CP30), and to reconstitute functionally active "30S" particles from free 16S RNA, CP30, and split proteins from 30S (SP30). It has been shown that neither yeast 16S ribosomal RNA nor rat liver 18S ribosomal RNA can replace coli 16S RNA in the reconstitution of "30S" particles. Degraded coli 16S RNA or degraded coli 23S ribosomal RNA are also ineffective. It has also been shown that core proteins from 50S ribosomal particles (CP50) cannot replace CP30 proteins. Thus, the specificity of the ribosomal RNA and the core proteins has been demonstrated. The efficient reconstitution of functional 30S ribosomal particles in the present system indicates that the entire information for the correct assembly of the ribosomal particles is contained in the structure of their molecular components, and not in some other nonribosomal factors. Materials and Methods. -The following buffer-salt mixtures were used: TMA I (10M Tris-HCl, pH 7.8, 10-2 Al MgCl2, 3 X 10M-2 7l NH4Cl, 6 X 10-3 M mercaptoethanol) TMA II (same as TMA I, but concentration of MgCl2 is 3 X 10-4 M); PMK I (5 X 10-3M H3P04 neutralized to pH 7.4 with KOH, 1.7 X 10-2M MgCl2, 3 X 10-3M CaCl2