Reaction of some macrolide antibiotics with the ribosome. Labeling of the binding site components

Abstract

Radioactive carbomycin A, niddamycin, tylosin, and spiramycin, but not erythromycin, can be covalently bound to Escherichia coli ribosomes by incubation at 37 degrees C. The incorporation of radioactivity into the particles is inhibited by SH- and activated double bond containing compounds but not by amino groups, suggesting that the reactions may take place by addition to the double bond present in the reactive antibiotics. This thermic reaction must be different from the photoreaction described for some of these macrolides [Tejedor, F., & Ballesta, J. P. G. (1985) Biochemistry 24, 467-472] since tylosin, which is not photoincorporated, is thermically bound to ribosomes. Most of the radioactivity is incorporated into the ribosomal proteins. Two-dimensional gel electrophoresis of proteins labeled by carbomycin A, niddamycin, and tylosin indicates that about 40% of the radioactivity is bound to protein L27; the rest is distributed among several other proteins such as L8, L2, and S12, to differing extents depending on the drug used. These results indicate, in accordance with previous data, that protein L27 plays an important role in the macrolide binding site, confirming that these drugs bind near the peptidyl transferase center of the ribosome.

Stepwise Binding of Tylosin and Erythromycin to Escherichia coli Ribosomes, Characterized by Kinetic and Footprinting Analysis

“…Post-translocation complex of poly(U)-programmed ribosomes, complex C, carrying tRNA Phe and Ac[ 3 H]Phe-tRNA at the E-and P-sites, respectively, was prepared in buffer A (100 mM Tris/HCl, pH 7.2, 4.5 mM Mg(CH 3 COO) 2 , 150 mM NH 4 Cl, and 6 mM 2-mercaptoethanol) and purified according to Dinos et al (31). Whenever required, 100 M spermine or a mixture of 50 M spermine and 2 mM spermidine was also included in buffer A.…”

“…Binding of Antibiotics and Probing of Complexes-Complex C at 100 nM was incubated alone or with antibiotics (I) at concentration equal to 50 ϫ K i in 100 l of buffer B (HEPES-KOH, pH 7.2, 4.5 mM Mg(CH 3 COO) 2 , 150 mM NH 4 Cl, 5 mM dithiothreitol) at 25°C, either for 10 s or for 8 ϫ t1 ⁄ 2 min, dependent on whether the encounter complex CI or the final complex C*I was desired to be probed, respectively. The term t1 ⁄2 , which represents the half-life for the attainment of equilibrium between complex C and the drug, was calculated through the relationship,…”

“…1) are representatives of the 14-and 16-membered lactone ring macrolides, respectively, both of which have been proposed to inhibit protein synthesis by binding to the large ribosomal subunit in a ratio of one molecule per ribosome (1,2). Erythromycin has only modest activity against Gram-negative enterobacteria (e.g.…”

“…E. coli TA531 cells that lack chromosomal rrn alleles but contain wild-type or mutant pKK3535 plasmids expressing wild-type or mutated 23 S rRNA (U2609C or U754A), respectively, were kindly provided by Prof. A. S. Mankin (University of Illinois) and used as a source of ribosomes in studying the effect of certain mutations on the mechanism of drug binding to E. coli ribosomes. Prior to their use, ribosomes were activated in buffer containing 20 mM Mg(CH 3 COO) 2 and 150 mM NH 4 Cl by incubation for 20 min at 42°C. Samples were then cooled at 0°C and the Mg 2ϩ concentration was normalized to 4.5 mM.…”

Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex.

“…However, immuno electron microscopic studies indicated that the proteins listed above are scattered over a large fraction of the ribosome surface (6) and clearly cannot all be in the vicinity of the tRNA 3' terminus. The diversity of proteins labeled no doubt derives, at least in part, from the size of the reactive substituents, as the crosslinks they establish are typically [10][11][12][13][14][15][16][17][18][19][20] A in length and may therefore extend to ribosomal components at a considerable distance from the site of tRNA binding.…”