Initiation Factor‐Dependent Binding of MS2 RNA to 30‐S Ribosomes and the Recycling of IF‐3

1 The binding of washed 30‐S ribosomal subunits of Escherichia coli to MS2 RNA in the absence of fMet‐tRNA requires the presence of the initiation factor IF‐3. No binding is observed with IF‐1, IF‐2 or a combination of these two initiation factors. 2 The IF‐3‐dependent binding is stimulated about two‐fold by IF‐2. IF‐1 has no effect in this respect. Optimal binding occurs at about 7 mM Mg2+. 3 Upon incubation of [35S]IF‐3, MS2 [3H]RNA and 30‐S subunits, complexes are formed which contain the three components in a 1:1:1 ratio. These ternary complexes have a sedimentation coefficient of about 40 S and a buoyant density in CsCl of about 1.74 g/ml. 4 The ternary complexes formed in the absence of IF‐2 and IF‐1 are rather labile and readily dissociate into MS2 RNA and IF‐3‐containing ribosomes. Their half‐life at 0°C is about 40 min. In the presence of IF‐2 and IF‐1, complexes are formed which remain stable for at least 6 h. Unwashed native 30‐S subunits also form stable complexes with MS2 RNA. 5 Binding of washed 30‐S subunits to unfolded MS2 RNA (MS2 RNA treated with formaldehyde) does not require initiation factors. Complexes containing more than one ribosomal particle per messenger can be formed. Attachment of fMet‐tRNA to these complexes requires IF‐2 and IF‐1, but is optimal in the presence of all three initiation factors. 6 Release of [35S]IF‐3 from the MS2 RNA · 30‐S subunit complexes occurs upon attachment of fMet‐tRNA. In the case of unwashed native 30‐S ribosomal subunits, recycling of IF‐3 occurs at the same stage as was demonstrated by a direct determination of the stoichiometry of unlabeled IF‐3 on the ribosomal complexes.