Antibiotic inhibition of RNA catalysis: neomycin B binds to the catalytic core of the td group I intron displacing essential metal ions1

Article No. mb982035

J. Mol. Biol. (1998) 282, 557±569

Antibiotic Inhibition of RNA Catalysis: Neomycin B Binds to the Catalytic Core of the td Group I Intron Displacing Essential Metal Ions I. Hoch1, C. Berens1, E. Westhof2 and R. Schroeder1* 1

Institute of Microbiology and Genetics, Vienna Biocenter Dr Bohrgasse 9 A-1030, Vienna, Austria 2

Institut de Biologie MoleÂculaire et Cellulaire du CNRS, F-67084, Strasbourg France

The aminoglycoside antibiotic neomycin B induces misreading of the genetic code during translation and inhibits several ribozymes. The self-splicing group I intron derived from the T4 phage thymidylate synthase (td) gene is one of these. Here we report how neomycin B binds to the intron RNA inhibiting splicing in vitro. Footprinting experiments identi®ed two major regions of protection by neomycin B: one in the internal loop between the stems P4 and P5 and the other in the catalytic core close to the G-binding site. Mutational analyses de®ned the latter as the inhibitory site. Splicing inhibition is strongly dependent on pH and Mg2‡ concentration, suggesting electrostatic interactions and competition with divalent metal ions. Fe2‡-induced hydroxyl radical (Fe-OH.) cleavage of the RNA backbone was used to monitor neomycin-mediated changes in the proximity of the metal ions. Neomycin B protected several positions in the catalytic core from Fe-OH. cleavage, suggesting that metal ions are displaced in the presence of the antibiotic. Mutation of the bulged nucleotide in the P7 stem, a position which is strongly protected by neomycin B from Fe-OH. cleavage and which has been proposed to be involved in binding an essential metal ion, renders splicing resistant to neomycin. These results allowed the docking of neomycin to the core of the group I intron in the 3D model. # 1998 Academic Press

*Corresponding author

Keywords: antibiotics; catalytic RNA; group I introns; metal ions; neomycin B

Introduction Neomycin B, an aminoglycoside antibiotic, has long been known to interfere with prokaryotic protein synthesis (Cundliffe, 1981, 1990; Davies et al., 1965; Tanaka, 1982). Both mutations and modi®cations leading to neomycin resistance as well as footprints located at the 16 S rRNA decoding site suggest an interaction of neomycin with the ribosomal RNA rather than with ribosomal proteins (Noller, 1991). Neomycin induces misreading of the genetic code, probably by interfering with the binding af®nity of non-cognate tRNAs to the decoding site (Karimi & Ehrenberg, 1996; Powers & Noller, 1994). The ribosomal target site of neomycin B is the 16 S rRNA 1400 to 1500 region, which has been clearly demonstrated by dissecting this domain from a small RNA of 27 nucleotides. This small subdomain of the 16 S rRNA was proE-mail address of the corresponding author: [email protected] 0022±2836/98/380557±13 $30.00/0

tected from chemical modi®cation by neomycin at the same positions as in the context of the 30 S subunit (Purohit & Stern, 1994). The NMR solution structure of this model RNA complexed with the aminoglycoside paromomycin has recently been solved, demonstrating how aminoglycosides can be accommodated by RNA (Fourmy et al., 1996). Several catalytic RNAs such as the self-splicing group I introns (von Ahsen et al., 1991), the hammerhead (Stage et al., 1995) and the Hepatitis Delta Virus (HDV; Rogers et al., 1996) ribozymes are inhibited by neomycin B. In addition to the above mentioned ribozymes, neomycin B inhibits HIV replication by binding to the Rev responsive element (RRE) and thereby preventing the Rev protein from binding to the viral RNA (Zapp et al., 1993). The hammerhead ribozyme is inhibited by neomycin B with a Ki of 13.5 mM (Stage et al., 1995). From the Mg2‡ and the pH dependence of inhibition it was proposed that protonated amino groups of neomycin undergo electrostatic inter# 1998 Academic Press

558 actions with the ribozyme (Clouet-d'Orval et al., 1995). A similar pH dependence of neomycin inhibition was reported for the ribozyme derived from the human HDV, where neomycin was proposed to interfere with catalysis by displacing essential metal ions (Rogers et al., 1996). Group I intron splicing is inhibited by several aminoglycosides such as neomycin B, gentamicin, 5-epi-sisomycin and tobramycin. They are strong non-competitive inhibitors with inhibitory constants in the high nanomolar to low micromolar range. The spectrum of inhibition differs between the ribosome and the group I intron. For example, kanamycin A and hygromycin, which are strong inhibitors of translation are very weak inhibitors of splicing. For splicing inhibition, amino groups on the antibiotic generally increase inhibition, whereas hydroxyl groups lower inhibitory ef®ciency (von Ahsen et al., 1992). A structure function analysis with deoxygenated aminoglycosides, which are more potent inhibitors of the hammerhead ribozyme than their corresponding analogues containing hydroxyl groups, clearly demonstrated that hydroxyl groups can affect the pKa values of neighbouring amino groups and that the basicity of these amino groups strongly determines the af®nity of the aminoglycoside to the RNA (Wang & Tor, 1997). RNA can be considered as a target for therapy and the detailed understanding of the mode of action of RNA-binding drugs should aid the rational design of novel therapeutics (Park et al., 1996). Understanding the principles underlying recognition and the mode of binding of aminoglycosides by RNA is currently a subject of intense analyses (Lato et al., 1995; Wallis et al., 1995; Wang & Rando, 1995; Famulok & HuÈttenhofer, 1996; Fourmy et al., 1996; Hendrix et al., 1997; Recht et al., 1996; Wang & Tor, 1997; Wang et al., 1997). The self-splicing group I intron td from the T4 phage was used as a model system for studying the mode of action of neomycin B. Footprinting analysis of neomycin B with the closely related sunY intron had previously revealed several bases in the core of the intron which were protected from chemical modi®cation in the presence of the antibiotic (von Ahsen & Noller, 1993). We mutated the protected bases and measured the effects of the mutations on the td ribozyme's sensitivity towards neomycin B. The in¯uence of Mg2‡ and pH on neomycin B inhibition as well as interactions with the RNA backbone were analysed. Iodine cleavage of phosphorothioate-substituted RNA was used, in the absence and presence of neomycin B, to detect changes in the accessibility of the RNA backbone due to antibiotic binding. A method using Fe2‡generated hydroxyl radicals was applied to measure the in¯uence of neomycin B on the binding of metal ions to the intron core. Taken together, these data allowed docking of neomycin into the 3D model of the td group I intron (Jaeger et al., 1993; Lehnert et al., 1996; Streicher et al., 1996). A mode of binding and inhibition is proposed

Splicing Inhibition by Neomycin B

which reveals structural principles for neomycin B/RNA interactions.

Results Mutation of neomycin footprinting sites Several bases in the group I intron core had previously been shown to be protected from chemical modi®cation by neomycin B (von Ahsen & Noller, 1993). To analyse the role of these bases in neomycin inhibition, we constructed a set of point mutations in the td intron. Figure 1A shows the mutations analysed in this study. Mutations at positions C56 and G938 did not result in severe splicing de®ciency in vitro. At 10 mM MgCl2, the mutant introns spliced with an activity similar to that of the wild-type (data not shown). The G871C:C946G:G1016A triple mutant requires high concentrations of ATP as the cofactor to splice ef®ciently. This is in good agreement with the effect of the mutation in the Tetrahymena ribozyme (Been & Perrotta, 1991). A very sensitive assay for thymidylate synthase activity and thus for ef®cient splicing of its intron is the in vivo phenotype on media lacking thymine (Belfort et al., 1987). Wild-type td
P62 and derived mutants were transformed into a thymine-de®cient Escherichia coli strain and growth was monitored on thymine-de®cient, trimethoprim and full media (Figure 1B). The mutants in J8/7 and J5/4 grew well on the minimal medium and only the C56G mutant showed an intermediate phenotype on trimethoprim-containing media, indicating that these mutations did not cause major splicing defects. The other mutants used in this work (C870U; C871C:C946G:G1016A and G871A:C946U (Michel et al., 1989) are splicing de®cient in vivo. Therefore, they cannot grow in the absence of thymine while they are resistant to trimethoprim. The mutations have only minor effects on the intron's sensitivity towards neomycin B The in¯uence of Mg2‡ on neomycin inhibition of the wild-type was tested. At suboptimal Mg2‡ concentrations (<3 mM), the intron is hypersensitive to neomycin B. However, between 5 and 10 mM Mg2‡, differences in neomycin sensitivity were negligible and within the error range (Figure 2). Thus, all the in vitro splicing assays were performed at 10 mM Mg2‡ to take into account the increased Mg2‡ requirement of several mutants. To obtain comparable splicing ef®ciencies for mutants and wild-type, GTP concentrations also had to be adjusted. As the required amounts of cofactor were signi®cantly higher for some of the mutants, we tested the in¯uence of high GTP on neomycin inhibition of wild-type splicing. As shown in Table 1, the differences between the Ki values at 2.5 and 100 mM GTP were insigni®cant. The rate limiting step in splicing of the td intron is exon ligation (second step), which is monitored in these assays.

Splicing Inhibition by Neomycin B

559

Figure 1. A, Secondary structure of the T4 phagederived td intron (adapted from Cech et al. (1994) with XRNA). Filled arrows indicate positions and nature of mutations, open arrows indicate 50 and 30 splice sites (50 ss and 30 ss). The G-binding site is surrounded by an open square. P1 to P9.2 designate the stems, J8/7 is the single-stranded junction between P8 and P7. The G871C:C946G:G1016A construct is a triple mutant. B, In vivo analysis-plating phenotypes. Constructs were plated on selective media: thy‡, non-selective medium; thyÿ, minimal medium lacking thymine; TTM, minimal medium supplemented with thymine and trimethoprim. wt, wild-type; CGA, mutant G871C:C946G:G1016A; AU, mutant G871A:C946U; C870U, G938C, C56A, C56G, C56U, single mutants.

560

Splicing Inhibition by Neomycin B

Figure 2. Magnesium dependence of inhibition of the td splicing reaction by neomycin B. v0/vi ˆ ratio of splicing activity (exon ligation) in the absence and presence of neomycin.

This step is not dependent on guanosine. In all mutants, exon ligation was still the rate limiting step. The sensitivity of the mutant introns to neomycin B was compared with that of the wild-type. A representative splicing reaction of the mutant in J8/7 (G938C) in the absence and presence of 0.25 mM neomycin B is shown in Figure 3. Table 1 indicates the Ki values for neomycin for the wildtype as well as for the mutants in J8/7, J5/4 and the G-binding site. The mutations had only minor effects on neomycin B sensitivity. These results clearly demonstrate that the mutations neither disrupt nor interfere with essential contacts between the antibiotic and the intron RNA. Thus, we conclude that the base protections observed previously result from indirect contacts or from direct contacts with non-inhibitory neomycin molecule(s). To probe changes in the accessibility of the backbone due to antibiotic binding we used the method developed by Eckstein and co-workers (Heidenreich et al., 1993; Schatz et al., 1991). Phosphorothioates were randomly incorporated into the Table 1. Inhibition constants (Ki) of wild-type and mutants for neomycin B Mutant Wild-type Wild-type G938C C56A C56G C56U G871C : C946G : G1016A C870U

GTP (mM)

Ki (mM)

2.5 100 2.5 50 5 25 1000a 100

0.17  0.07 0.11  0.05 0.11  0.03 0.48  0.24 0.36  0.2 0.26  0.08 0.36  0.14 54  14

mM GTP ˆ concentration of GTP at which Ki was determined. a The mutant G871C : C946G : G1016A requires ATP as a cofactor for splicing instead of GTP.

Figure 3. Splicing inhibition of the mutant G938C by neomycin B. Pre-RNA was incubated with 30 mM GTP for 20 to 160 seconds at 37 C in the absence and presence of 0.25 mM neomycin B, respectively. Bands are labelled as follows: pre, precursor RNA; I-E2, intron-30 exon; lin. I, linear intron; E1-E2, ligated exons; E1, 50 exon.

RNA during in vitro transcription and the renatured RNA was subsequently cleaved with iodine in the absence and presence of neomycin B. Backbone footprints of neomycin B accumulate in the P4/P6 domain with the joining regions J5/4 and J6/7 being most prominently protected (data not shown and Figure 5B). Protections were only detected at antibiotic concentrations much higher than the inhibitory concentrations. We must therefore assume that the inhibitory neomycin molecule(s) does not protect any phosphate or, due to some inexplicable reason, it is only capable of protecting the backbone from iodine at much higher concentrations. pH dependence of splicing inhibition by neomycin B Several amino groups of neomycin B are protonated at pH 7 and deprotonated between pH 7 and pH 9 (Figure 4A; Botto & Coxon, 1983). For the hammerhead ribozyme, the pH dependence of inhibition suggested that at least three of the ®ve positively charged ammonium groups of neomycin B are critical for inhibition (Clouet-d'Orval et al., 1995). We measured inhibition of wild-type td splicing by neomycin B at different pH values, ranging from pH 7 to pH 8.5. Within this range, splicing activity is not affected by pH changes (Herschlag & Khosla, 1994). As shown in Figure 4B, inhibition is strongly pH dependent. At pH 7.5 inhibition is still effective, whereas at pH 7.8 the ef®ciency of neomycin is signi®cantly reduced and at pH 8

561

Splicing Inhibition by Neomycin B

activity at pH 7.8, and the 60 amino group only deprotonates at pH 8.6. The importance of this group is demonstrated by paromomycin, which contains, as the sole difference to neomycin B, an OH group at position 60 , and kanamycin C, which also contains a hydroxyl group at this position. Paromomycin inhibits splicing at 36 mM (data not shown) and kanamycin C is inactive at 5 mM (von Ahsen et al., 1991). Fe2‡-generated hydroxyl radical cleavage in the presence of neomycin B

Figure 4. A, Structure of neomycin B with the respective pKa values of its amino groups (Botto & Coxon, 1983). B, pH dependence of inhibition of the td splicing reaction by neomycin B. %lig. exons, percentage of the product (ligated exons) relative to total amount of RNA.

splicing is nearly unaffected by the antibiotic. Thus, protonated ammonium groups are most probably essential for binding of neomycin B to the intron RNA. The 20 and 2000 amino groups deprotonate around pH 7.6. The importance of the 20 amino group had been demonstrated earlier by a structure function analysis: kanamycin B, with a 20 amino group inhibits splicing at 10 mM and kanamycin A, with a 20 hydroxyl group as the only difference, does not inhibit at 5 mM (von Ahsen et al., 1991, 1992). The 2000 amino group is not expected to be essential as ring IV is variable and not conserved among aminoglycosides. The role of the 60 amino group cannot be determined by pH variation, as the antibiotic loses its inhibitory

To test whether divalent metal ion binding sites in the group I intron are potential targets for the antibiotic, we employed a recently developed method to probe for the surroundings of metal ions (Berens et al., 1998). Fe2‡, which is similar to Mg2‡ in both size and coordination geometry (Brown, 1988) can, in the absence of EDTA, structurally or functionally substitute for Mg2‡, as has been shown for protein-ligand complexes (see, e.g., Ettner et al., 1995; Lykke-Andersen et al., 1997; Zaychikov et al., 1996). In the presence of sodium ascorbate and hydrogen peroxide, Fe2‡ generates hydroxyl radicals which can cleave the phosphodiester backbone (Latham & Cech, 1989; Tullius & Dombroski, 1985; Wang & Cech, 1992). Incubation of group I intron RNAs with Fe2‡ revealed several conserved strong cleavage sites in the intron core (J5/4, J6/7, J8/7 and P7) as well as other cleavage sites in the peripheral extensions (Berens et al., 1998). To determine if these sites are affected by neomycin B, we cleaved the RNA with Fe2‡ in the presence of increasing concentrations of this antibiotic. As speci®city controls we used kanamycin A and spermidine. A typical cleavage gel is shown in Figure 5A. In the presence of neomycin B, several sites change in cleavage intensity. They are located in J5/4 (U72; not shown), in J6/7 (U867), in P7 (C870) and J8/7 (G938, U940, and U942). Signals in L7.1 (G883, G884) and J7.2/3 (A907, A908) stem from a single peripheral metal ion binding site (Berens et al., 1998). In Figure 5B all protections are summarized. Of these, only A908 shows an increase in cleavage intensity, all others are cleaved less ef®ciently. In addition, cleavage at four nucleotides is affected by both neomycin B and kanamycin A. G864 and A868 are cleaved more strongly, U889 and A890 less intensively. Protection from Fe2‡-OH radical cleavage is detected at 5 mM neomycin, which is higher than the Ki of inhibition (0.17 mM). This is due to the assay conditions necessary for these experiments: the RNA concentration is 1 mM and thus the ratio RNA to antibiotic is 1:5, which is also the ratio observed for inhibition. In the presence of spermidine (1 to 500 mM), these patterns do not change (data not shown), indicating that non-speci®c interactions with the backbone do not affect the Fe2‡-generated cleavage sites. Several sites affected by neomycin B are close to the previously proposed A and B metal ion binding sites (Streicher et al., 1996). The protection

562

Splicing Inhibition by Neomycin B

of positions U72, U867 and C870 is a clear indication that the A-site metal ion is displaced from its original position. The same is true for the B-site ion as deduced from protections in J8/7. Mutating the bulged nucleotide in P7 (C870U) results in resistance towards neomycin B The bulged nucleotide in the P7 stem is semiconserved and introns with C and A at this position are active under physiological conditions. Mutation of this nucleotide in the td intron from C to U or G results in an increased requirement for Mg2‡ and for the cofactor guanosine (Schroeder et al., 1991). Metal-induced cleavage of the RNA backbone resulted in the proposal that the N3 position of C was involved in the coordination of the essential

A-site metal ion (Streicher et al., 1996). Since this position was strongly protected from Fe2‡-induced cleavage in the presence of neomycin, we analysed the sensitivity of the C870U variant towards neomycin B. Wild-type and the C870U constructs were analysed at identical conditions (10 mM MgCl2 and 100 mM GTP) and their sensitivity towards neomycin was compared. The Ki of the mutant for neomycin B was found to be 54 mM, while it is 0.11 mM for the wild-type. Thus the C870U mutant is 500-fold more resistant to neomycin B than the wild-type (Table 1 and Figure 6A and B). The sensitivity of this mutant towards paromomycin was also tested and found to be 46 mM (data not shown), while the Ki for the wild-type is 36 mM. This indicates that the mutant cannot discriminate between neomycin and paromomycin, suggesting

Figure 5 (legend opposite)

Splicing Inhibition by Neomycin B

563

Figure 5. A, Probing for metal-binding sites in the td intron in the absence and presence of antibiotics. Intron RNA was cleaved with Fe2‡, sodium ascorbate and hydrogen peroxide after incubation with increasing amounts of neomycin B (Neo B) or kanamycin A (Kan A). G is a RNase T1 sequencing lane, AH an alkaline hydrolysis ladder. Several secondary structure elements of td are indicated on the left. On the right, nucleotide positions are indicated where the Fe2‡-mediated cleavage is strongly decreased in the presence of neomycin B, arrows indicate subtle effects. B, Secondary structure of the td intron. Positions protected from iodine cleavage by neomycin B are indicated by ®lled arrows, different sizes of arrows re¯ect different grades of protection. Filled circles designate nucleotide positions which are cleaved less ef®ciently, open circles nucleotide positions which are cleaved more ef®ciently by Fe2‡-generated hydroxyl radicals in the presence of neomycin B (see A). Numbering of nucleotides and designation of secondary structure elements as in Figure 1A.

that the 60 amino group of ring I of neomycin B, which is missing in paromomycin, interacts with the bulged nucleotide in the core of the intron. This interaction provides a ®rst constraint for modelling neomycin B into the intron core.

Discussion Chemical protection experiments with DMS and kethoxal, which probe for base contacts (von

Ahsen & Noller, 1993) and with iodine cleavage of phosphorothioate-substituted RNA, which probes for backbone contacts, revealed several footprints in the presence of neomycin B scattered over the 2D structure of the intron. Some of these positions converge to de®ned regions in space in the threedimensional model available for the subgroup to which the td intron belongs (Jaeger et al., 1993; Lehnert et al., 1996; Streicher et al., 1996). Two major sites of protection by neomycin B were

564

Figure 6. Splicing inhibition of wild-type and mutant C870U by neomycin B. A, Wild-type precursor RNA was renatured as described in Materials and Methods and splicing was initiated by adding 100 mM GTP, and time points up to 120 seconds were taken. Increase of product (ligated exons) was plotted versus time and the plot was corrected for hydrolysis background. Slopes indicate % exon ligation per second. B, Mutant C870U precursor RNA was treated as the wild-type, time points were taken up to 150 seconds in the presence of 0, 0.1 and 40 mM neomycin B, respectively. Formation of ligated exons was plotted as in A.

detected, one in the internal loop between the stems P4 and P5 and the other one surrounding the G-binding core. Mutational analyses in J4/5 and J8/7 suggest that no interactions essential for inhibition of exon ligation occur between the protected bases and the antibiotic. In contrast, mutation of a single position in the core (C870U) resulted in neomycin resistance, indicating that binding of neomycin B to this site is responsible for inhibition. In previous work on the modelling of aminoglycosides into the hammerhead ribozyme (Hermann & Westhof, 1998), molecular dynamics simulations of neomycin B in aqueous and neutralized solutions were performed. In that report, it was

Splicing Inhibition by Neomycin B

shown that the intramolecular distances between the ammonium groups in the antibiotic matched the inter-ionic distances between the magnesium ions in the ribozyme as deduced from X-ray crystallography (Scott et al., 1996). Most importantly, the ammonium groups at positions 1 and 20 were shown to replace the magnesium ions closest to the cleavage site. We applied the same strategy to the td intron and docked eight different conformers of neomycin B (extracted from the MD simulations and representing the main conformational families) to the G-binding site so that the ions at sites A and B were displaced by ammonium groups on neomycin B rings I and II. Using this strategy, it was never possible to cover the two major protected regions with one neomycin B molecule. We thus concluded that there are at least two binding sites for neomycin B on the td intron. One major site of protection lies in the internal loop between the stems P4 and P5 and was not modelled precisely owing to the lack of data. Also, protection occurs at antibiotic concentrations which are much higher than for inhibition. The asymmetric P4/P5 internal loop has been shown to be involved in docking the P1 stem harbouring the 50 splice site (Cech et al., 1994; Michel & Westhof, 1990). There are several examples of asymmetric internal loops which bind aminoglycoside antibiotics: both the Rev responsive element of HIV and the decoding site of the 16 S rRNA are asymmetric internal loops, where the loops are closed by non-canonical base-pairs. In both cases the antibiotic contacts the deep groove (Fourmy et al., 1996; Zapp et al., 1993). An in vitro selection for neomycin binding RNAs resulted in the isolation of stem-loops with helical irregularities, which open the deep groove (Wallis et al., 1995). Most probably, neomycin B sits on the deep groove side of the P4/P5 loop on the opposite side of the P1 recognition region. This loop should thus not be the inhibitory binding site. The functional similarity between the P4/P5 domain of group I introns and the decoding site of 16 S rRNA A-site is remarkable, as both RNAs bind a short RNA helix, most probably via the 20 OH groups of the RNA backbone (Fourmy et al., 1996; Pyle et al., 1992; Schroeder et al., 1993; Strobel & Cech, 1994; Strobel et al., 1997). How neomycin binds to the internal loop between P4 and P5 remains to be analysed, but in analogy to the decoding process, aminoglycosides should not inhibit the docking of the helix, but rather affect the binding speci®city (Karimi & Ehrenberg, 1994, 1996). Modelling neomycin B into the td intron core The second major site of protection surrounds the G-binding core and since a mutation at a single position (C870) in the core results in resistance, we conclude that this is the inhibitory site. The docking of neomycin B to the G-binding core could be achieved by all conformers in such a way (Figure 7A, B and C) that (i) the two magnesium

Splicing Inhibition by Neomycin B

565 ions are displaced by the ammonium groups of rings I and II, and (ii) the G-cofactor binding is not hindered (non-competitive inhibition). With all conformers tested, rings III and IV point towards the solvent where the substrate is expected to bind. The conformers distinguish themselves by rotations about the glycosyl bonds between rings I and II. Thus, while ammonium 1 on ring II always replaced magnesium ion B, magnesium ion A was alternatively replaced by the ammonium group 20 or 60 on ring I. Thus, those aminoglycoside derivatives lacking an ammonium group at either 20 or 60 are less effective inhibitors, since only a subset of conformers present in solution can simultaneously replace both magnesium ions. Single point mutations have previously been reported to be responsible for resistance to aminoglycosides. A single G to A mutation in the decoding site of the prokaryotic ribosome renders translation insensitive to aminoglycosides (De Stasio et al., 1989). Interestingly, an A to G mutation in the human mitochondrial 12 S rRNA was reported to create a high af®nity binding site for aminoglycosides, causing a hereditary form of aminoglycoside-induced deafness (Hamasaki & Rando, 1997). Our results clearly demonstrate that metal ions are displaced by the antibiotic, as neomycin B protects several distinct positions from cleavage. Since divalent metal ions are crucial for catalysis of most ribozymes, displacing them is a very ef®cient way of inhibition of catalysis. For group I intron splicing, at least two metal ions are essential. Metal ion-induced cleavage of the RNA backbone lead to the structural model, where two metals surround the 50 splice site. These ions have been designated A and B according to the Steitz & Steitz model (Steitz & Steitz, 1993; Streicher et al., 1996). The requirement for both ions has been demonstrated by chemical interference studies (Piccirilli et al., 1993; Weinstein et al., 1997) and by kinetic experiments (McConnell et al., 1997). The metal ion A was proposed to contact the bulged nucleotide in P7 (Streicher et al., 1996) and the G-cofactor (SjoÈgren et al., 1997). The metal ion B is in position to coordinate the 30 bridging oxygen at the 50 splice site (Piccirilli et al., 1993). Neomycin, as modelled into the core of the td intron, displaces both ions;

Figure 7. Views of neomycin B docked into the catalytic core of the intron. The domain P4-P6 is in green and domain P7 is in purple (see Figure 1A). The G-cofactor is in orange. The G-C to which it binds is in cyan. The bulge C is in purple. The antibiotic neomycin is colourcoded so that nitrogen atoms are blue, oxygen red and

carbon white. The yellow spheres represent the proposed positions for two Mg2‡ critical for catalysis (Streicher et al., 1996); ion A is close to the bulge C and ion B at the bottom of the picture. Ring I of neomycin B (here nitrogen 20 ) is close to ion A and ring II (nitrogen 1) to ion B. For clarity, metal ions are shown together with neomycin B; in reality the metals are displaced in the presence of the antibiotic. A, View down the G-binding site. B, View after a rotation about a horizontal axis showing rings III and IV above the G-cofactor. C, Space®lling drawing of the complex between the td intron and neomycin B (in red, at the centre). In blue, domain P2; in yellow domain P9; and in orange domain P7. The P4-P6 domain is at the back.

566 the 20 amino group of neomycin ring I displaces ion A and the 1 amino group of ring II displaces ion B (Figures 4A, 7A and B). Alternatively, since rotation can occur around the glycosidic bond between rings I and II, the 60 amino group of ring I can displace ion A. The modelling data are supported by the resistance of the bulge mutant (C870U) to neomycin B and by the inability of this mutant to discriminate between neomycin B and paromomycin. The modelling data further suggest that the N3 position of C870 is close to the 3 amino group of ring II, such that these two groups together with the 60 amino group form an equilateral triangle. However, we could not test this interaction due to the lack of an aminoglycoside antibiotic with a 3 hydroxyl group in ring II. Inhibition of ribozyme catalysis by neomycin B might in general occur by metal ion displacement. All ribozymes which have been tested to date for sensitivity towards aminoglycosides are indeed inhibited (Schroeder & von Ahsen, 1996). There is only one ribozyme where no divalent metal ion appears to be required for the catalytic step: the hairpin ribozyme (Chowrira & Burke, 1991; Hampel & Cowan, 1997; Nesbit et al., 1997; Young et al., 1997). The very recent observation that this ribozyme is resistant to neomycin B (Zachary Taylor & John Burke, personal communication) is in total agreement with our observation that metal ion-binding sites are the speci®c targets of aminoglycosides in ribozymes.

Materials and Methods Bacterial strains and plasmids The E.coli strain used for in vivo tests in this study is a thymine-de®cient derivative of C600 (Fÿ, supE44, thi-1, thr-1, leuB6, lacY1, tonA21,
thyA). In vitro splicing experiments were performed with a truncated version of the T4 phage-derived thymidylate synthase (td) gene, containing 79 nt of exon I, 265 nt of the intron (
P6-2) and 21 nt of exon II (Galloway Salvo et al., 1990; Schroeder et al., 1991). For iodine and Fenton cleavage experiments, the ribozyme construct tdL-7 was used. This construct lacks the ®rst seven bases of the intron (Heuer et al., 1991).

Media and growth conditions Growth medium was TBYE (1% (w/v) Bactotryptone, 0.5% (w/v) NaCl, 0.5% (w/v) yeast extract and 50 mg/ ml thymine). Minimal medium supplemented with Casamino acids (Belfort et al., 1983) but lacking thymine (thyÿ medium) was used to select for the Td‡ (TS‡) phenotype. TTM, a minimal medium supplemented with thymine (50 mg/ml) and trimethoprim (20 mg/ml), was used to screen for the Tdÿ (TSÿ) phenotype, since only cells lacking TS activity are able to grow on media containing the folate analogue trimethoprim. In addition, all media contained 100 mg/l ampicillin. Cells were grown at 37 C.

Splicing Inhibition by Neomycin B In vitro mutagenesis For introduction of mutation C56X, a mixed oligonucleotide (50 -TCTACTAGAGAGXTTCCCCGTTTAG-30 , where X ˆ A, T or C) was used. Deoxyuridine-substituted single-stranded DNA was isolated following the protocol of Kunkel et al. (1987). Annealing of the phosphorylated oligonucleotide to single-stranded DNA, elongation and ligation were as described by Williamson et al. (1989). After transformation, the colonies were screened on TTM for the Tdÿ phenotype. Mutants G871A:C946U designated AU and G938C were kindly provided by T. Hirsch and mutant G871C:C946G:G1016A by Herbert Wank. The C870U construct was reported previously (Schroeder et al., 1991). RNA preparation Precursor RNA (preRNA) of the T4 phage-derived thymidylate synthase (td) intron was transcribed in vitro from plasmid td
P6-2 (Schroeder et al., 1991) with [a-35S]CTP and puri®ed according to the procedure described by Streicher et al. (1993). Ribozyme construct tdL-7 was prepared by transcription under hydrolysis conditions (pH 8.0, 15 mM MgCl2). To obtain tdL-7 precursor RNA for iodine backbone cleavage, transcription mixtures were doped with one of the phosphorothioate analogues at a ratio of 10%. Transcriptions were performed under hydrolysis conditions, but otherwise as described by Streicher et al. (1993). In vitro splicing assays For renaturation, the gel-puri®ed precursor RNA was incubated with splicing buffer containing magnesium for two minutes at 56 C. Subsequently, a zero value was taken, the reaction was started with GTP and time points were taken. Reactions were stopped by precipitation with 0.3 M NaOAc/EtOH and reaction products were separated on 5% (w/v) denaturing acrylamide gels. In the case of mutant G871C:C946G:G1016A, to avoid hydrolysis, precursor RNA was not renatured after gel puri®cation. For this mutant, the cofactor which initiates the reaction is ATP instead of GTP. The GTP requirements of all other mutants were tested and GTP concentrations were chosen in a way so as to obtain splicing activities similar to that of the wild-type at 2.5 mM GTP. Unless otherwise indicated, all splicing and inhibition experiments were performed in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 0.4 mM spermidine with 20 ng of precursor RNA. When the pH dependence of splicing inhibition was determined, incubation buffer was as described above but contained 50 mM Hepes (pH 7.0, 7.2, 7.5, 8.0 or 8.5). Precursor RNA was renatured as described above, antibiotic was added to the corresponding reactions and a zero value was taken. Subsequently, the reaction was started with GTP and incubated for two minutes at 37 C, while a control without GTP was incubated in the same way to correct for hydrolysis. Each experiment was done in triplicate. Gels were scanned on a PhosphorImager and analysed as described below. Ki determination Splicing time courses of wild-type and mutants were performed as described above in the absence and pre-

567

Splicing Inhibition by Neomycin B sence of various neomycin B concentrations and gels were scanned on a PhosphorImager. Increase per second of ligated exons, expressed as percentage of the sum of precursor and all product bands, was monitored as initial velocities in the absence (v0) and presence (vi) of antibiotic. The values of v0/vi were plotted against antibiotic concentrations, and Ki was determined as the concentration at which v0/vi equals 2, which means that the initial velocity is halved by the antibiotic. Iodine cleavage of phosphorothioate substituted precursor RNA Phosphorothioate substituted RNA was 50 -endlabelled with [g-33P]ATP and preincubated with splice buffer for three minutes at 56 C. Subsequently, antibiotic was added and allowed to bind for two minutes at room temperature. Iodine was added to a ®nal concentration of 1 mM and the reaction was incubated at room temperature for one minute, stopped by the addition of four volumes stop solution (2.5 mM EDTA, 0.1 mg/ml yeast tRNA), precipitated with three volumes of 0.3 M NaOAc/EtOH and cleavage products were separated on 6% denaturing acrylamide gels. Each experiment was repeated at least thrice, bands that turned out to be reproducibly protected were visually preselected and quanti®ed. The total radioactivity of each lane was measured and the percentage of each band was determined. By expressing the intensity of one band as the percentage of the total radioactivity of a given lane, slight loading differences between lanes are neutralized. Protection was de®ned as signi®cant when the decrease was at least 30%. Fenton cleavage of antibiotic-treated RNA 50 -[g-32P]- and 30 -[a-32P]-labelling of RNA was as described (Lingner & Keller, 1993; Sambrook et al., 1989). Sequencing ladders were generated by limited hydrolysis with RNase T1 and NaHCO3 (Donis-Keller et al., 1977). For the Fe2‡-mediated cleavage of antibiotic-treated RNA, 1 ml tdL-7 RNA (6 pmol; approximately 30,000 cpm) was added to 1 ml 6 native cleavage buffer (1 NCB is 25 mM Mops-KOH (pH 7.0), 3 mM MgCl2), incubated for two minutes at 56 C and for three minutes at room temperature. 1 ml of either neomycin B, kanamycin A or spermidine (®nal concentrations ranging from 0.5 to 500 mM) was added and the reaction incubated for two minutes at room temperature. 1 ml of 1.5 mM FeCl2 was then added and incubated for one minute before adding 1 ml each of 15 mM sodium ascorbate and 15 mM H2O2 to initiate the reaction. The reaction was stopped after 60 seconds by adding thiourea to a ®nal concentration of 150 mM. The RNA was precipitated with ethanol, dissolved in RNA-loading buffer and loaded onto 6% to 8% denaturing polyacrylamide gels. Modelling The aminoglycosides were manually docked into the 3D model of the td intron using FRODO (Jones, 1978). The conformers used for the aminoglycosides were derived by molecular dynamics simulations using the program AMBER 4.1 (Pearlman et al., 1994) as described by Herman & Westhof (1998). The 3D views were made using DRAWNA (Massire et al., 1994).

Acknowledgements We are grateful to John Burke and Zachary Taylor for communicating results prior to publication. We thank Herbert Wank for the CGA mutant, Thomas Hirsch for the G938C mutant, Norbert Polacek for the Ki of the CGA mutant, Thomas Hermann for the MD conformers of aminoglycosides, Bryn Weiser for the XRNA program and Norbert Polacek, Barbara Streicher and Mary G. Wallis for comments on the manuscript. E.W. thanks the Institut Universitaire de France for support. This work is funded by the Austrian Science Foundation (FWF) grant no. P11362 and P11999 to R.S. and by the European Community TMR program, grant no. FMRX-CT97-0154 to R.S. and E.W.

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Edited by M. Yaniv (Received 6 April 1998; received in revised form 25 June 1998; accepted 25 June 1998)