Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli.
We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.
Walleczek
J
J
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, FRG.
Martin
T
T
Redl
B
B
Stöffler-Meilicke
M
M
Stöffler
G
G
D003196:Computer Graphics; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004852:Epoxy Compounds / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D008961:Models, Structural; D008970:Molecular Weight; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D003432:Cross-Linking Reagents; D004852:Epoxy Compounds; D012269:Ribosomal Proteins; C007366:diepoxybutane