Binding of the Initiation Factor IF‐3 to Escherichia coli Ribosomes and MS2 RNA

The binding of IF‐3 to 70‐S, 50‐S and 30‐S ribosomes and subunits of Escherichia coli and to MS2 RNA was studied employing Sephadex G‐200 chromatography and sucrose‐gradient centrifugation to recover the complex formed. In the latter case, reaction mixtures were fixed with 0.25% glutaraldehyde prior to centrifugation. Total reaction mixtures were also analyzed by polyacrylamide‐gel electrophoresis. Highly purified IF‐3, either unlabeled or labeled with 35 S was employed and consistent results were obtained with both factor preparations. Binding of IF‐3 to 30‐S subunits was independent of the Mg 2+ concentration in the range 1–14 mM. The binding to 50‐S subunits and MS2 RNA was counteracted by increasing the Mg 2+ concentration in the same range. Binding at 0°C and at 37°C occurred to the same extent both with 30‐S and 50‐S ribosomal subunits. The number of IF‐3 molecules bound per 30‐S subunit approached unity, that bound per 50‐S subunit exceeded unity when the IF‐3 input was sufficiently raised. At low factor/ribosome input ratios IF‐3 binds preferentially to 30‐S particles. When the ratio is increased binding to 50‐S subunits and 70‐S ribosomes also occurs. The IF‐3‐mediated dissociation of 70‐S ribosomes is strongly inhibited by adding increasing amounts of 30‐S subunits. At all Mg 2+ concentrations studied the relative extent of binding can be expressed as: 30‐S > 70‐S, 50‐S > MS2 RNA. Binding to all types of ribosomes and subunits and to MS2 RNA is not stimulated by the addition of IF‐1 and IF‐2. The latter factors stabilize the 30‐S · IF‐3 complexes, however.