Cross-linking of initiation factor IF-2 to Escherichia coli 30 S ribosomal proteins with dimethylsuberimidate.

Abstract

International audienceThe 30 S ribosomal proteins near the binding site for initiation factor IF-2 in Escherichia coli were identified by allowing complexes of 30 S subunits, [32P]phosphoryl initiation factor IF-2 and nonradioactive initiation factors IF-1 and IF-3, to react with the protein cross-linking reagent dimethylsuberimidate. Noncross-linked initiation factors were removed by centrifugation of the complexes in buffer containing a high salt concentration; the protein was extracted from the pelleted particles; and cross-linked species containing initiation factor IF-2 and ribosomal proteins were partially purified by column chromatography on Sephadex G-75. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against 20 individual 30 S ribosomal proteins S1, S2, S11, S12, S13, S14, and S19 was interpreted to mean that initiation factor IF-2 was present in covalent cross-linked complexes containing those proteins. The results imply that these 30 S ribosomal proteins are near the binding site for initiation factor IF-2.The 30 S ribosomal proteins near the binding site for initiation factor IF-2 in Escherichia coli were identified by allowing complexes of 30 S subunits, [32P]phosphoryl initiation factor IF-2 and nonradioactive initiation factors IF-1 and IF-3, to react with the protein cross-linking reagent dimethylsuberimidate. Noncross-linked initiation factors were removed by centrifugation of the complexes in buffer containing a high salt concentration; the protein was extracted from the pelleted particles; and cross-linked species containing initiation factor IF-2 and ribosomal proteins were partially purified by column chromatography on Sephadex G-75. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against 20 individual 30 S ribosomal proteins S1, S2, S11, S12, S13, S14, and S19 was interpreted to mean that initiation factor IF-2 was present in covalent cross-linked complexes containing those proteins. The results imply that these 30 S ribosomal proteins are near the binding site for initiation factor IF-2