Direct cross-links between initiation factors 1, 2, and 3 and ribosomal proteins promoted by 2-iminothiolane.
Complexes were prepared containing 30S ribosomal subunits from Escherichia coli and the three initiation factors IF1, IF2, and IF3. In different experiments, each of the factors was radiolabeled with the others unlabeled. The complexes were allowed to react with 2-iminothiolane and then oxidized to promote the formation of intermolecular disulfide bonds, some of which were between factors and ribosomal proteins. Each of the labeled factors becomes covalently cross-linked to the complex as judged by its failure to dissociate when centrifuged in a sucrose gradient containing a high salt concentration. Proteins from the complexes were extracted and analyzed on two-dimensional polyacrylamide gels by nonequilibrium isoelectric focusing and sodium dodecyl sulfate gel electrophoresis. Spots corresponding to cross-linked dimers that contained initiation factors, as indicated on autoradiographs, were cut out and analyzed further. The following cross-linked dimers between factors and ribosomal proteins were identified: IF1-S12, IF1-S18, IF2-S13, IF3-S7, IF3-S11, IF3-S13, and IF3-S19. Cross-links between factors IF1-IF2 and IF3-IF2 were also identified. A model integrating these findings with others on the protein topography of the ribosome is presented.
D001665:Binding Sites; D003432:Cross-Linking Reagents; D004926:Escherichia coli; D007096:Imidoesters; D007467:Iodoproteins / Q000032:analysis; D010084:Oxidation-Reduction; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure
D003432:Cross-Linking Reagents; D007096:Imidoesters; D007467:Iodoproteins; D010448:Peptide Initiation Factors; D012269:Ribosomal Proteins; C005905:methyl 4-mercaptobutyrimidate