The EMBO Journal vol. 1 0 no. 10 pp. 3099 - 3103, 1991 Interaction of 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 , 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 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- 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 -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 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 , 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 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.

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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 ; 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. stem-loop of P-site tRNA is bound during its interaction Apramycin and neamine, which both cause miscoding and with mRNA. Direct evidence for the proximity of two of inhibit translocation (Perzynski et al., 1979; Delcuve et al., these loops comes from intramolecular crosslinking of 16S 1987), give footprints in the same tightly constrained region rRNA in 30S ribosomal subunits between bases located of the 16S rRNA secondary structure that is located at the approximately at positions 695 and 794 or 799, by treatment base of the penultimate stem. This highly conserved region with bis-(2-chloroethyl)methylamine (Atmadja et al., 1986). is the site where certain bases are protected by the anticodon If the protection of bases by edeine, pactamycin and stem-loop region of A-site-bound tRNA (Moazed and kasugamycin is the result of direct contact between these Noller, 1986, 1990), accounting for the miscoding effects drugs and the protected bases, then our data can be taken of these drugs. Their footprints are, therefore, similar to as evidence supporting the mutual proximity of these three those of the neomycin-related antibiotics neomycin, structural features: because of the relatively small molecular kanamycin, gentamicin and paromomycin, and of dimensions of the antibiotics, positions 693, 794-795 and hygromycin (Moazed and Noller, 1987a), whose effects are 926 must be relatively close to each other for any two of also to cause miscoding and inhibit translocation. A simple the RNA sites to be simultaneously in contact with the same explanation for their miscoding effects is that they perturb 3101 J.Woodcock et al.

those displayed by streptomycin. Both drugs affect trans- Table I. Protection of bases in 16S rRNA by antibiotics and tRNA lational accuracy and inhibit the initiation of protein synthesis Ligand Protected bases (Davies et al., 1988). Myomycin and streptomycin pheno- G693 A794 C795 G926 typically suppress the same range of nonsense and missense mutations of E. coli. Furthermore, single amino acid changes P-site tRNA + + + + in ribosomal protein S12 can cause cross resistance to both Edeine + + + + drugs (Davies et al., 1988). These close similarities provide Pactamycin + - + strong evidence that myomycin and streptomycin share the (N 1,N7) same ribosomal target site-a conclusion reinforced by the Kasugamycin - + E + studies of Montandon et al. (1986) which demonstrate that A1408 G1491 G1494 a C to U base change at position 912 in E. coli 16S rRNA (N7) (N7) gives streptomycin resistant strains that are cross resistant to myomycin (Davies et al., 1988). Nevertheless, although A-site tRNA 4 - + the primary sites protected within 16S rRNA by streptomycin Neomycins + + + are in the 915 region, we find that myomycin protects no Hygromycin E + + bases within this region (data not shown). Instead, it gives Apramycin + + + very weak protection of A1408-a result which suggests that Neamine i + + myomycin, like neomycin and related aminoglycosides, Myomycin 4- might perturb A-site codon - anticodon interaction in some Symbols indicate (+) protection or (±) weak protection from or (E) way. It may be that structural differences between strepto- enhancement of chemical modification of the designated bases. Ni and mycin and myomycin, although allowing an identical N7 refer to the site of modification of guanine bases that is protected. inhibitory action, cause the drugs to bind to the same Data are from Moazed and Noller (1986, 1990) (A-site and P-site on ribosomes but at different contact tRNA); Moazed and Noller (1987a) (edeine, neomycins and functional domain hygromycin), and this paper (pactamycin, kasugamycin, apramycin, points. However, we note that the protection of bases neamine and myomycin). 911-915 by streptomycin is incomplete, even when its binding to the ribosome is saturated (D.Moazed, unpublished; T.Powers and H.F.Noller, submitted). Similarly, protection of A1408 by myomycin is also incomplete at high drug concentrations, suggesting that the observed protections by both drugs are indirect, and not due to direct contact with 16S rRNA. If this is the case, we conclude that the true binding sites for streptomycin and myomycin have yet to be identified. G693 > An important unsolved problem is whether the observed protection of bases by antibiotics and tRNA (Table I) results A 794 from direct ligand -rRNA contact, or from ligand-induced conformational changes in 16S rRNA. All four of the previously reported protections by edeine mimicked four P- site tRNA protections (Moazed and Noller, 1987a, 1990), a result that could be interpreted in terms of a conformational change induced by either edeine or tRNA. However, the results of the present study argue against such a simple interpretation. Thus, two of the four sites (G693 and C795) are protected by pactamycin, whereas this drug has no effect on the reactivity of the other two sites; therefore, there would have to be two conformational changes. The finding that, unlike for tRNA and edeine, pactamycin protects not only the NI position of G693 but also the N7 position, introduces Fig. 3. Model for the folding of 16S rRNA in 30S ribosomal subunits further complications, and would require that the putative (Stem et al., 1988b), showing the locations of bases that are protected conformational change is somehow different in the case of in common by P-site-bound tRNA and by edeine, pactamycin and this drug. Furthermore, pactamycin protects C795 without kasugamycin (cf. Table I). Residues G693, A794, C795 and G926 are affecting the adjacent base A794 (which is protected by all protected by tRNA as well as by edeine. Pactamycin protects G693 and C795, while kasugamycin protects A794 and G926, and enhances kasugamycin, edeine and tRNA). Here again, the results are C795. more simply accounted for in terms of direct contact than by multiple, independent conformational changes. Such an the site in the 30S ribosomal subunit where anticodon -codon interpretation is consistent with our current understanding recognition takes place, perhaps by strengthening non- of 16S rRNA structure. specific interactions between A-site tRNA and the ribosome. Another question, raised implicitly by these studies, is the Such a mechanism might also help to explain their inhibitory role of ribosomal proteins in the interactions of antibiotics effect on translocation. with ribosomes. Antibiotic resistance is well known to be The results obtained for myomycin are surprising and conferred by mutations in r-proteins. In addition to the intriguing. This antibiotic shares certain structural features possibility that the proteins may contribute all or part of with kasugamycin, streptothricin and streptomycin, but the certain drug-binding sites, their role(s) could, alternatively, inhibitory actions of myomycin are apparently identical to be in the modulation of the detailed conformation of rRNA 3102 Antibiotics and 16S rRNA

(Allen and Noller, 1989; Stern et al. 1989). In at least one Stern,S., Moazed,D. and Noller,H.F. (1988a) Methods Enzymol., 164, case, that of thiostrepton (Cundliffe, 1990), it has been shown 481 -489. that the rRNA itself is capable of binding the drug in the Stem,S., Weiser,B. and Noller,H.F. (1988b) J. Mol. Biol., 204, 447-481. absence of protein. It remains to be seen how far this will Stern,S.,244, 783-790.Powers,T., Changchien,L.-M. and Noller,H.F. (1989) Science, extend to the many other antibiotics that interact with Tai,P.-C., Wallace,B.J. and Davis,B.D. (1973) Biochemistry, 12, 616-620. ribosomes. Received on May 7, 1991; revised on June 17, 1991 Materials and methods Antibiotics were obtained from the following sources: kasugamycin, Sigma; pactamycin, The Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute; apramycin, Lilly Research Laboratories, Indianapolis; neamine, Upjohn Company, Kalamazoo, MI; myomycin, Parke-Davis, Ann Arbor, MI. Ribosomes were prepared from E. coli MRE600 as described previously (Moazed and Noller, 1989). Antibiotics were bound to 100 pmol of 70S ribosomes in 100 Id of 80 mM potassium cacodylate (pH 7.2) 20 mM MgCl2, 100 mM NH4Cl, 1 mM DTT, 0.5 mM EDTA for 30 min at 37°C and then for 10 min on ice. Antibiotic concentrations were: kasugamycin, 200 1M; myomycin, 50 zM; apramycin, 100 /tM; neamine, 100 /M; pactamycin 100 itM. Chemical modification was performed by addition of dimethyl sulfate or kethoxal followed by incubation at 37°C for 10 min, as described previously (Moazed and Noller, 1987a). The rRNA was then isolated by phenol extraction, precipitated with ethanol and the sites of chemical modification identified by primer extension as detailed previously (Stem et al., 1988a).

Acknowledgements J.W. and M.C. gratefully acknowledge the financial support of the Science and Engineering Research Council, the Nuffield Foundation and the Wellcome Trust. D.M. and H.F.N. were supported by NIH grant No. GM-17129. We thank Lilly Research Laboratories, the Drug Synthesis and Chemistry Branch of the National Cancer Institute, Upjohn Laboratories, Parke-Davis, Inc., E.De Stasio and A.E.Dahlberg for gifts of antibiotics. We thank Bryn Weiser for computer graphics drawings.

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