Identification of proteins at the subunit interface of the Escherichia coli ribosome by cross-linking with dimethyl 3,3'-dithiobis(propionimidate).
The 70S ribosomes of Escherichia coli were treated with dimethyl 3,3'-dithiobis(propionimidate). Under conditions where 40% of the lysine epsilon-amino groups became modified, about 50% of the ribosomes became resistant to dissociation into 30S and 50S subunits when analyzed in the absence of reducing agents on sucrose gradients containing low magnesium concentrations. Dissociation took place in the presence of reducing agents, indicating that the bifunctional reagent had reacted with proteins from both subunits. Proteins were extracted from purified cross-linked 70S ribosomes by using conditions to preclude disulfide interchange. Disulfide-linked protein complexes and non-cross-linked proteins were first fractionated by electrophoresis in polyacrylamide/urea gels at pH 5.5. The proteins from sequential slices of the urea gel were analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Monomeric proteins derived from cross-linked dimers appeared below the diagonal of non-cross-linked proteins since the second electrophoresis but not the first is run under reducing conditions to cleave the cross-linked species. Final identification of the constituent proteins in each dimer was made by radioiodination of the cross-linked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of nonradioactive marker 70S protein. The identification of 11 cross-linked protein dimers which contained one protein from each of the two ribosomal subunits is described. We conclude that the proteins in these cross-linked pairs are located in the regions of contact between the two subunits, i.e., at the "subunit interface".
D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis*; D007096:Imidoesters; D046911:Macromolecular Substances; D008970:Molecular Weight; D012269:Ribosomal Proteins / Q000032:analysis*; D012270:Ribosomes / Q000032:analysis*; D014508:Urea
D003432:Cross-Linking Reagents; D007096:Imidoesters; D046911:Macromolecular Substances; D012269:Ribosomal Proteins; C007471:dimethyl dithiobispropionimidate; D014508:Urea