Affinity labeling of the virginiamycin S binding site on bacterial ribosome.
Virginiamycin S (VS, a type B synergimycin) inhibits peptide bond synthesis in vitro and in vivo. The attachment of virginiamycin S to the large ribosomal subunit (50S) is competitively inhibited by erythromycin (Ery, a macrolide) and enhanced by virginiamycin M (VM, a type A synergimycin). We have previously shown, by fluorescence energy transfer measurements, that virginiamycin S binds at the base of the central protuberance of 50S, the putative location of peptidyltransferase domain [Di Giambattista et al. (1986) Biochemistry 25, 3540-3547]. In the present work, the ribosomal protein components at the virginiamycin S binding site were affinity labeled by the N-hydroxysuccinimide ester derivative (HSE) of this antibiotic. Evidence has been provided for (a) the association constant of HSE-ribosome complex formation being similar to that of native virginiamycin S, (b) HSE binding to ribosomes being antagonized by erythromycin and enhanced by virginiamycin M, and (c) a specific linkage of HSE with a single region of 50S, with virtually no fixation to 30S. After dissociation of covalent ribosome-HSE complexes, the resulting ribosomal proteins have been fractionated by electrophoresis and blotted to nitrocellulose, and the HSE-binding proteins have been detected by an immunoenzymometric procedure. More than 80% of label was present within a double spot corresponding to proteins L18 and L22, whose Rfs were modified by the affinity-labeling reagent. It is concluded that these proteins are components of the peptidyltransferase domain of bacterial ribosomes, for which a topographical model, including the available literature data, is proposed.
Di Giambattista
M
M
Unit of Microbiology and Genetics, ICP, Medical School, University of Louvain, Brussels, Belgium.
Nyssen
E
E
Pecher
A
A
Cocito
C
C
D000345:Affinity Labels; D000917:Antibody Formation; D001483:Base Sequence; D001665:Binding Sites; D007136:Immunoglobulins / Q000276:immunology; D008969:Molecular Sequence Data; D010458:Peptidyl Transferases / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry* / Q000187:drug effects; D013388:Succinimides; D014769:Virginiamycin / Q000737:chemistry* / Q000276:immunology
D000345:Affinity Labels; D007136:Immunoglobulins; D012269:Ribosomal Proteins; D013388:Succinimides; D014769:Virginiamycin; D010458:Peptidyl Transferases; C001426:N-hydroxysuccinimide