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Bases in 16S Ribosomal RNA

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Bases in 16S Ribosomal RNA The EMBO Journal vol. 1 0 no. 10 pp. 3099 - 3103, 1991 Interaction of antibiotics with A- and P-site-specific bases in 16S ribosomal RNA Joanna Woodcock, Danesh Moazed1"3, 16S and/or 23S rRNA from chemical probes when they bind Michael Cannon, Julian Davies2 and to ribosomes, each producing a characteristic footprint on Harry F.Noller1 the rRNA (Moazed and Noller, 1987a,b). The protected nucleotides are, in most cases, identical with or located Department of Biochemistry, Division of Biomolecular Sciences, adjacent to bases that have been directly implicated in those King's College, London WC2R 2LS, UK, 'Sinsheimer Laboratories, functional processes known by University of California at Santa Cruz, CA 95064, USA and 2Unit6 de to be affected the antibiotic Genie Microbiologique, Institut Pasteur, 75724 Paris, France in question. Accordingly, it has been suggested that the mode(s) of action of such drugs may be to interfere directly 3Present address: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143, with the function of highly conserved sites in rRNA (Moazed USA and Noller, 1987a,b; Noller et al., 1990). Communicated In this study, we report the effects of several additional by J.Davies 30S subunit-specific antibiotics: kasugamycin, pactamycin, apramycin, neamine and myomycin. Kasugamycin and pactamycin are known to inhibit translational initiation We have studied the interactions of the antibiotics (Cohen et al., 1969; Okuyama et al., 1971; Tai,P.-C. et al., apramycin, kasugamycin, myomycin, neamine and 1973), while both apramycin and neamine not only increase pactamycin with 16S rRNA by chemical probing of the frequency of translational errors but also inhibit drug-ribosome complexes. Kasugamycin and pacta- translocation (Delcuve et al., 1978; Perzynski et al., 1979). mycin, which are believed to affect translational Myomycin has a limited structural resemblance to strepto- initiation, protect bases in common with P-site-bound mycin. Nevertheless, these two aminoglycoside-amino- tRNA. While kasugamycin protects A794 and G926, and cyclitol antibiotics apparently share an identical mode of causes enhanced reactivity of C795, pactamycin protects action, inducing misreading and inhibiting translational G693 and C795. All four of these bases were previously initiation in cell-free protein synthesizing systems (Davies shown to be protected by P-site tRNA or by edeine, et al., 1988). We show that kasugamycin and pactamycin another P-site inhibitor. Apramycin and neamine, which protect P-site-specific bases, which may explain how they both induce miscoding and inhibit translocation, protect block initiation. Apramycin and neamine protect bases in A1408, G1419 and G1494, as was also found earlier for or closely adjacent to tRNA A-site-protected sites on neomycin, gentamicin, kanamycin and paromomycin. ribosomes, in keeping with their abilities to increase A1408 and G1494 were previously shown to be protected miscoding. Interestingly, myomycin gives only very weak by A-site tRNA. Surprisingly, myomycin fails to give protection of a single base within 16S rRNA, and fails to strong protection of any bases in 16S rRNA, in spite of protect any bases in common with streptomycin, despite having an apparently identical target site and mode of the apparently identical inhibitory actions of these two action to streptomycin, which protects several bases in antibiotics. the 915 region. Instead, myomycin gives only weak protection of A1408. These results suggest that the binding site(s) of streptomycin and myomycin have yet Results to be identified. The different antibiotics were bound to Escherichia coli 70S Key words: antibiotic/chemical probing/rRNA/ribosome ribosomes at concentrations where they are known to exert their effects on translation, and probed with the single-strand- specific RNA probes kethoxal and dimethyl sulfate. The sites of chemical modification and protection by the drugs were Introduction identified by primer extension. All of the antibiotic-dependent A wide range of antibiotics act by inhibiting protein protections are shown in Figure 1; for each of the panels, synthesis, and the majority of these drugs interact directly lane 1 shows the modification pattern for drug-free with ribosomes (Cundliffe, 1981). In early studies on ribosomes, while the other numbered lanes show the effects antibiotic-resistance mutations that affected ribosomes, of the various antibiotics. Since pactamycin had to be attention was drawn to the ribosomal proteins as possible dissolved in ethanol, lane 7 shows the results of probing target sites for antibiotic interaction. More recently, ribosomes treated with ethanol alone (5%) as a control however, resistance mutations for many antibiotics have been against possible effects caused by the solvent. found that result in alterations of ribosomal RNA (rRNA), Pactamycin and kasugamycin, two inhibitors of trans- raising the possibility that such antibiotics may actually lational initiation, both protect bases in 16S rRNA that are interact with rRNA, perhaps exclusively (De Stasio et al., also protected by P-site binding of tRNA (Moazed and 1988). Indeed, many antibiotics, with the notable exceptions Noller, 1990). Pactamycin protects G693 at both its Nl and of puromycin (Moazed and Noller, 1987b) and sparsomycin N7 positions (as inferred by protection from both kethoxal (Moazed and Noller, 1991), protect specific nucleotides in and DMS modification), and C795 at N3. Kasugamycin ©) Oxford University Press 3099 J.Woodcock et al. .. .~.,e~?'.~~.S ~~~~~~~~~~~~~...... r.Ig... ,d ir.: io .: Fig. 1. Autoradiographs showing protection of bases in 16S rRNA caused by binding of antibiotics to 70S ribosomes. A and G are dideoxy sequencing lanes. Lane K, unmodified 70S ribosomes. Lanes 1-7 are 70S ribosomes modified in the presence or absence of the various ligands. Lane 1, no antibiotic; lane 2, kasugamycin; lane 3, myomycin; lane 4, apramycin; lane 5, neamine; lane 6, pactamycin; lane 7, ethanol (5 per cent). Chernical modification was done with kethoxal (A and D) and dimethyl sulfate (B, C, E and F); aniline-induced strand scission was performed for DMS-modified samples in B and F, to identify N7 methylation of guanine. protects A794 at NI and G926 at NI and, in addition, causes -Edeine, pactamycin and kasugamycin, all of which inhibit enhanced reactivity of C795 at its N3 position. translational initiation, protect sites that are also protected Apramycin and neamine, both of which induce miscoding by P-site tRNA. Since initiation involves binding of initiator and inhibit translocation, protect the NI of A1408 and the tRNA to the 30S P-site, our footprinting results can account N7 positions of G1491 and G1494. These nucleotides are in a straightforward way for the mode of action of these three positioned at the base of the penultimate stem of the drugs. While edeine protects four of the strongly protected secondary structure of 16S rRNA (Figure 2A) in and around P-site bases (G693, A794, C795 and G926), the other two the location of the tRNA A-site footprint (Moazed and drugs each affect a different subset. Pactamycin protects Noller, 1990). G693, although in a manner that is clearly distinguishable The results obtained with myomycin were very unexpected from that of edeine. Thus, whereas the latter protects the since this antibiotic is regarded as having a mode of NI position of G693, pactamycin protects both positions NI inhibitory action that is identical to that of streptomycin and N7. In addition, although pactamycin protects C795, (Davies et al., 1988). Myomycin gives only a very weak, it has no protective effects on either A794 or G926. In although reproducible, protection of a single base-AI408. contrast, kasugamycin protects A794 and G926, and causes We have failed to detect strong protection or enhancement enhanced DMS reactivity at C794, but has no effect on of any bases in 16S rRNA by this drug. The data for G693. Thus, these three antibiotics that are all known to protection of bases in 16S rRNA by antibiotics and tRNA affect initiation of protein synthesis, a process that involves are s-immarized in Table I. the function of the ribosomal P site, interact with the 16S rRNA P site in exquisitely different ways. These Discussion observations may be attributed to the fact that the three drugs have distinctly different chemical structures (Cundliffe, Figure 2 summarizes the chemical footprinting results for 1981), which are reflected in inhibitory actions that have A- and P-site tRNA on 16S rRNA (Moazed and Noller, evolved independently to affect the same functional target 1990) and compares them with the corresponding footprints site on ribosomes. obtained from binding antibiotics in this paper as well as In spite of the wide separation of these four P-site bases from earlier work (Moazed and Noller, 1987a). The two in the secondary structure, they are believed to be relatively sets of data show a clear correlation. close to each other as a result of the tertiary folding of 16S 3100 Antibiotics and 16S rRNA A p Fig. 2. Locations of antibiotic-protected bases in 16S rRNA (Moazed and Noller, 1987a; this work), compared with bases protected by tRNA bound to the ribosomal A- and P-sites (Moazed and Noller, 1990). Vertical arrows indicate enhancement of reactivity. On the left are shown tRNA- protected bases, and on the right are the corresponding antibiotic protections. Apr, apramycin; Ede, edeine; Hyg, hygromycin; Ksg, kasugamycin; Myo, myomycin; Nea, neamine; Neos, neomycin and related aminoglycosides; Pct, pactamycin. rRNA in the ribosome. Figure 3 shows the location of the drug molecule. In the model of Stern et al. (1988b), the 690, 790 and 930 region stems in the three-dimensional distances are 24 A (G693/A794) and 25 A (G693/G926 and model of 16S rRNA proposed by Stern et al. (1988b). In A794/G926), respectively. Within the uncertainty of the the latter study, it was proposed that these three stems model, these distances are consistent with possible direct surround the cleft of the 30S subunit, where the anticodon contact between the antibiotics and the protected bases.
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