Abstract
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.
Highlights
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, [9ZP]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 crosslinked species containing initiation factor IF-2 and ribosomal proteins were partially purified by column chromatography on Sephadex G-75
Identification of the ribosomal proteins constituting the binding site for IF-2 by cross-linking requires that the initiation factor bind under the conditions employed for the reaction
Summary
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, [9ZP]phosphoryl initiation factor IF-2 and nonradioactive initiation factors IF-1 and IF-3, to react with the protein cross-linking reagent dimethylsuberimidate. A variety of methods have been used to determine the topography of the 21 proteins of the Escherichia coli 30 S ribosomal subunit (for reviews, see Sun et al [1] and Traut et al [2]) Previous studies in this laboratory [3,4,5] have emphasized the use of bifunctional protein cross-linking reagents for the identification of pairs or groups of neighboring proteins in the isolated ribosomal subunit. The work reported here deals with the identification of ribosomal proteins near the binding site for IF-2 on the 30 S subunit
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