Photo-Induced Labelling of Escherichia coli Ribosomes by a Tobramycin Analog

Abstract

An [3H]azidobenzyl derivative of tobramycin, a 4,6-disubstituted 2-deoxystreptamine aminoglycoside, has been synthesized, and its ability to label Escherichia coli 70-S ribosomes under photoactivation has been studied. Two concentrations of the photolabel, corresponding to the saturation of the two classes of tobramycin sites on the ribosomes, were used. The results show that, at high antibiotic concentrations which induce maximal misreading during protein synthesis, most of the ribosomal proteins are labelled. At low antibiotic concentration, which results in the saturation of the first-class sites, a few proteins of both subunits are labelled, including L6, S4, S 5, and, to a lesser extent, L2, L13 and S18. The 30-S subunit is, on the whole, labelled more efficiently than the 50-S subunit.In spite of the great similarity in structure of aminoglycosides, recent studies have suggested that there are differences in their mode of action at the level of the bacterial ribosomes. Whereas the facts seem clear as to the differences which exist between aminoglycosides containing the streptidine residue (streptomycin) and those containing 2-deoxystreptamine [I -51 the differences between the aminoglycosides derived from kanosamine (kanamycin, tobramycin) and those derived from garosamine (gentamicin, sisomicin) are seldom shown. It is clear, however, that these two families of closely related antibiotics behave differently in relation to the biosynthesis of proteins [I] and their binding on bacterial ribosomes [5, 71 (and Moukaddem, Tangy and Le Goffic, unpublished results).We have shown that tobramycin interacts on two classes of sites of the 70-S ribosome [4, 51: the first class, of high affinity (Kd = 0.2 pM), is saturated at a low concentration, which is also responsible for a significant inhibition of R17 mRNA translation [8, 91; the second class, of lower affinity (Kd = 10 pM), can accept a large number of antibiotic molecules. The high concentrations necessary to saturate this latter family of sites suggest that the conformational modifications which they induce on both the 50-S and the 3 0 4 subunits are responsible for the misreading. It should be noticed that such aminoglycoside concentrations also inhibit the dissociation of ribosomes into their subunits [lo].It seemed, therefore, of interest to identify the ribosomal proteins involved with these antibiotics in order to determine whether the latter have a specific action on protein synthesis or whether they simply modify the conformation of the ribosomes in a non-specific, yet efficient, way.In this communication, we present the results of affinity labelling experiments using Escherichia coli ribosomes, and a photoreactivable derivative of tobramycin.

Specificity of Aminoglycoside Binding to RNA Constructs Derived from the 16S rRNA Decoding Region and the HIV-RRE Activator Region

“…ART, an arginyl substituted tobramycin analog, was prepared for study because arginyl moieties have proved to interact strongly with RNA molecules through electrostatic interactions (Calnan et al, 1991). Regiospecific synthesis of tobramycin analogs could be readily achieved, because the 6′-primary amino group is selectively acylated as a consequence of being the sole unhindered primary amino group (Tangy et al, 1983;LeGoffic et al, 1979). Competitive binding experiments were carried out under the same conditions as described above for the competition of Rev 34-50 with Fl-Rev 34-50 .…”

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.…”

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

“…Some of the labeled proteins have been related also to the interaction site of other antibiotics that affect the initiation of protein synthesis. Thus, protein SI3 seems to be involved in streptomycin interaction (Leon-Rivera, 1981), proteins S4 and L13 in the tobramycin site (Tangy et al, 1983), proteins SI8 and S21 in pleuromutilin inhibition (Hogenauer et al, 1981), and protein S2 in kasugamycin resistance (Okuyama et al, 1974).…”