About the specificity of photoinduced affinity labeling of Escherichia coli ribosomes by dihydrorosaramicin, a macrolide related to erythromycin

Abstract

Photoactivation of the [3H]dihydrorosaramicin chromophore at a wavelength above 300 nm allows the covalent attachment of the macrolide antibiotic to the bacterial ribosome. Bidimensional electrophoresis shows that the radioactivityisniainly associated with proteins L1, L5, L6, L15, L18, L19, S1, S3, S4, S 5 and S9. When photoincorporation of the drug is conducted in the presence of puromycin as effector of ['H]dihydrorosaramicinbinding sites, a decrease in the labeling of most proteins is observed, except for LIX and Ll9, which are radiolabeled to a larger extent. These results allow us to speculate that L 18 and L 19 belong to the high-affinity binding site of rosaramicin antibiotic.Photoaffinity labeling has become an important method for identifying functional sites on the Escherichia coli ribosome [l] and antibiotics such as puromycin and chloramphenicol have been used as tools for providing information on the peptidyltransferase center [2 -41.Although it has been a quarter century since macrolide antibiotics were reported to block protein synthesis [S] the mechanism by which they accomplish this inhibition has remained unknown. Erythromycin, the major representative of the macrolide group, binds on the large subunit ofprokaryotic ribosomes [6] and, among other assumptions, could be considered as an inhibitor of the peptidyl residue near the peptidyltransferase [7]. In contrast to the large number of studies concerning the mode of action of the macrolides, very little is known about the macromolecules involved in the structure of their binding site.Rosaramicin (Fig. 1) is a 16-membered macrolide, whose activity is similar to that of erythromycin. This antibiotic possesses an aldehyde function, easily reducible to an alcohol group without loss of antibiotic activity; this led to the obtaining of a tritiated molecule, which was used to determine rosaramicin-binding parameters onto E. coli ribosomes, showing one high-affinity binding site (Kd = 0.16 x M) and multiple low-affinity binding sites [8, 91. Moreover, the antibiotic has an a,p-ethylenic y,S-epoxyketo system that offers a promising approach to photoactivated affinity labeling. This method, which eliminated the need for preparing reactive derivatives, has been recently used to label steroid-binding proteins and receptors [lo]. In a preliminary report [Ill we have shown that photoexcitement of ['H]dihydrorosaramicin allows its covalent attachment to E. coli ribosome proteins, mainly on L1, L5, L6 and S1. Recently components of the macrolide-binding site on the ribosome have been identified [ 121 using dihydrocarbomycin under different labeling conditions and showing L27 as the major labeled protein.

Affinity labeling of the virginiamycin S binding site on the bacterial ribosome

“…Affinity labeling by aminoacyl-tRNA at the ribosomal A site has pointed to proteins L2, L15, L16, and L27 and that with P site-bound aminoacyl-tRNA to L2 and L27 (Pellegrini & Cantor, 1977). Moreover, analysis of macrolide-resistant mutants and affinity labeling of ribosomal proteins by these antibiotics have shown the involvement of L27 (Tejedor & Ballesta, 1986), L18 (Siegrist et al, 1985), L22 and L4 (Wittmann et al, 1973;Pardo & Rosset, 1977;Arevalo et al, 1988), and L15 (Hummel et al, 1979;Arevalo et al, 1988). Two of the macrolide-binding L proteins correspond to those identified in the present work as virginiamycin S labeled proteins.…”

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

“…Moreover, mutations in protein S12 affect the binding of macrolides to ribosomes from erythromycin-resistant strains (Saltzman & Apirion, 1976). The location of both the peptidyl transferase center and the binding sites of several other antibiotics at the subunit interface of the ribosome has also been suggested by affinity labeling studies using different types of probes that in most cases also label proteins from the small subunit (Nicholson et al, 1982a,b;Pongs & Messer, 1976;Tangy et al, 1983; On the basis of affinity labeling results using the macrolide rosaramycin, proteins L18 and L19 have been suggested as components of its binding site (Siegrist et al, 1985). Protein LI8, as part of the complex with 5S RNA, has been located in the central protuberance of the 50S subunit (Stoffler-Meilicke et al, 1983) and therefore close to protein L27.…”

Βιοσυνθεση Του Πεπτιδικου Δεσμου Και Αναστολη Της Απο Ορισμενα Αντιβιοτικα

“…1.6.1. Μελέτες προσδέσεως ραδιενεργών αντιβιοτικών Έχουν ως στόχο, τον εντοπισμό εξειδικευμένης θέσεως προσδέσεως στο ριβόσωμα, και υπολογισμό αφ' ενός μεν της σταθεράς διαστάσεως του αντιστρεπτούς σχηματιζόμενου συμπλόκου, αφ' ετέρου δε την μοριακή αναλογία στο σχηματιζόμενο σύμπλοκο (Fernandez-Munoz et al, 1971;Harris & Pestka 1973;Siegrist et al, 1985).…”