Crosslinking of streptomycin to the 30S subunit of Escherichia coli with phenyldiglyoxal

Abstract

[3H]Dihydrostreptomycin was covalently linked to the 30S subunit of E. coli K12A19 with the bifunctional cross-linking reagent phenyldiglyoxal. The cross-linking was abolished under conditions that prevent the binding of streptomycin, which indicates that the cross-linking occurs at the specific binding site of streptomycin. The cross-linking involved 16S RNA and the ribosomal proteins S1, S5, S11 and S13, suggesting that the streptomycin binding site is located in the upper part of the 30S subunit, facing the 50S subunit. Unexpectedly, the same extent and pattern of cross-linking were observed with the 30S subunits from a streptomycin-resistant mutant. Streptomycin induces conformational changes in the ribosomes from sensitive bacteria but not from streptomycin-resistant mutants. This and the results in the present study suggest that the binding of streptomycin to streptomycin-sensitive ribosomes is a 2-step reaction wherein an initial loose interaction at the antibiotic binding site is followed by a conformational rearrangement of the ribosomal particle. The second step would tighten the association with streptomycin and cause interference with protein synthesis. That step would be lacking in streptomycin-resistant mutants.