STRUCTURE AND FUNCTION OF ESCHERICHIA COLI RIBOSOMES .6. MECHANISM OF ASSEMBLY OF 30-S RIBOSOMES STUDIED IN VITRO

Reconstitution of 30 s ribosomes from 16 s ribosomal RNA and a mixture of 30 s ribosomal proteins was studied. Several factors affecting the efficiency of the reconstitution were examined. It was found that there is a sharp optimum (I = 0.37) for the ionic strength of the reconstitution mixture. Magnesium ions must be present in the reconstitution mixture at a concentration of 0.01 m or higher (measured up to 0.03 m). With a 15-minute incubation at 40 °C, the maximum efficiency was obtained at pH values between 6.5 and 8.0 (at 40 °C). The reconstitution reaction was first-order with respect to formation of active 30 s ribosomes. Changes in the total concentration of the reactants do not affect the kinetics of reconstitution, expressed as a percentage of the final value. Thus the rate-limiting reaction is probably unimolecular. The rate of reconstitution is strongly dependent on the temperature of the incubation mixture. From the dependence of the rate constant on temperature, an Arrhenius activation energy of 37.8 kcal./mole has been obtained. Calculation has also given a positive value for the entropy of activation. Incubation of the reconstitution mixture at 0 °C does not produce 30 s ribosomes. The particles (presumed reconstitution intermediates, or RI particles) isolated by centrifugation from such reconstitution mixtures are deficient in several protein components (S proteins), which are recovered from the supernatant fraction after centrifugation. RI particles are functionally inactive. Neither restoration of their functional activity by S proteins nor efficient binding of S proteins to RI particles can take place at 0 °C, but do so only after incubation at higher temperatures. However, heating RI particles alone at 40 °C for 20 minutes produces particles (activated reconstitution intermediates, or RI* particles) which are now capable of binding S proteins at 0 °C and becoming functional 30 s ribosomes. Thus, the reconstitution takes place in step-wise fashion: 16 s RNA → +RI proteins RI particles → heating RI* particles → +S proteins 30 s ribosomes. The reaction RI particles → RI* particles is the ratelimiting unimolecular reaction, representing a structural rearrangement of RI particles which requires high activation energy. Experiments are also described in which formation of functionally active 30 s ribosomes from unfolded 30 s ribosomes was demonstrated. The kinetics of this refolding process under the conditions used has been shown to be almost identical to those of reconstitution of 30 s ribosomes from RNA and proteins. It is shown that the same intermediate, the RI particle, is involved in both reactions. © 1969.