antibiotic inhibition of rna catalysis: neomycin b … · antibiotic inhibition of rna catalysis:...

Antibiotic Inhibition of RNA Catalysis: Neomycin BBinds to the Catalytic Core of the td Group I IntronDisplacing Essential Metal Ions

I. Hoch1, C. Berens1, E. Westhof2 and R. Schroeder1*

1Institute of Microbiology andGenetics, Vienna BiocenterDr Bohrgasse 9A-1030, Vienna, Austria2Institut de BiologieMoleÂculaire et Cellulaire duCNRS, F-67084, StrasbourgFrance

The aminoglycoside antibiotic neomycin B induces misreading of the gen-etic code during translation and inhibits several ribozymes. The self-spli-cing 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 intronRNA inhibiting splicing in vitro. Footprinting experiments identi®ed twomajor regions of protection by neomycin B: one in the internal loopbetween the stems P4 and P5 and the other in the catalytic core close tothe G-binding site. Mutational analyses de®ned the latter as the inhibi-tory site. Splicing inhibition is strongly dependent on pH and Mg2� con-centration, suggesting electrostatic interactions and competition withdivalent metal ions. Fe2�-induced hydroxyl radical (Fe-OH.) cleavage ofthe RNA backbone was used to monitor neomycin-mediated changes inthe proximity of the metal ions. Neomycin B protected several positionsin the catalytic core from Fe-OH. cleavage, suggesting that metal ions aredisplaced in the presence of the antibiotic. Mutation of the bulged nucleo-tide in the P7 stem, a position which is strongly protected by neomycin Bfrom Fe-OH. cleavage and which has been proposed to be involved inbinding an essential metal ion, renders splicing resistant to neomycin.These results allowed the docking of neomycin to the core of the group Iintron in the 3D model.

# 1998 Academic Press

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

Introduction

Neomycin B, an aminoglycoside antibiotic, haslong been known to interfere with prokaryotic pro-tein synthesis (Cundliffe, 1981, 1990; Davies et al.,1965; Tanaka, 1982). Both mutations and modi®-cations leading to neomycin resistance as well asfootprints located at the 16 S rRNA decoding sitesuggest an interaction of neomycin with the riboso-mal RNA rather than with ribosomal proteins(Noller, 1991). Neomycin induces misreading ofthe genetic code, probably by interfering with thebinding af®nity of non-cognate tRNAs to thedecoding site (Karimi & Ehrenberg, 1996; Powers& Noller, 1994). The ribosomal target site of neo-mycin B is the 16 S rRNA 1400 to 1500 region,which has been clearly demonstrated by dissectingthis domain from a small RNA of 27 nucleotides.This small subdomain of the 16 S rRNA was pro-

tected from chemical modi®cation by neomycin atthe same positions as in the context of the 30 S sub-unit (Purohit & Stern, 1994). The NMR solutionstructure of this model RNA complexed with theaminoglycoside paromomycin has recently beensolved, demonstrating how aminoglycosides canbe accommodated by RNA (Fourmy et al., 1996).

Several catalytic RNAs such as the self-splicinggroup I introns (von Ahsen et al., 1991), the ham-merhead (Stage et al., 1995) and the Hepatitis DeltaVirus (HDV; Rogers et al., 1996) ribozymes areinhibited by neomycin B. In addition to the abovementioned ribozymes, neomycin B inhibits HIVreplication by binding to the Rev responsiveelement (RRE) and thereby preventing the Rev pro-tein from binding to the viral RNA (Zapp et al.,1993).

The hammerhead ribozyme is inhibited by neo-mycin B with a Ki of 13.5 mM (Stage et al., 1995).From the Mg2� and the pH dependence of inhi-bition it was proposed that protonated aminogroups of neomycin undergo electrostatic inter-

E-mail address of the corresponding author:[email protected]

Article No. mb982035 J. Mol. Biol. (1998) 282, 557±569

0022±2836/98/380557±13 $30.00/0 # 1998 Academic Press

actions with the ribozyme (Clouet-d'Orval et al.,1995). A similar pH dependence of neomycin inhi-bition was reported for the ribozyme derived fromthe human HDV, where neomycin was proposedto interfere with catalysis by displacing essentialmetal ions (Rogers et al., 1996).

Group I intron splicing is inhibited by severalaminoglycosides such as neomycin B, gentamicin,5-epi-sisomycin and tobramycin. They are strongnon-competitive inhibitors with inhibitory con-stants in the high nanomolar to low micromolarrange. The spectrum of inhibition differs betweenthe ribosome and the group I intron. For example,kanamycin A and hygromycin, which are stronginhibitors of translation are very weak inhibitors ofsplicing. For splicing inhibition, amino groups onthe antibiotic generally increase inhibition, whereashydroxyl groups lower inhibitory ef®ciency (vonAhsen et al., 1992). A structure function analysiswith deoxygenated aminoglycosides, which aremore potent inhibitors of the hammerhead ribo-zyme than their corresponding analogues contain-ing hydroxyl groups, clearly demonstrated thathydroxyl groups can affect the pKa values of neigh-bouring amino groups and that the basicity ofthese amino groups strongly determines the af®-nity of the aminoglycoside to the RNA (Wang &Tor, 1997).

RNA can be considered as a target for therapyand the detailed understanding of the mode ofaction of RNA-binding drugs should aid therational design of novel therapeutics (Park et al.,1996). Understanding the principles underlyingrecognition and the mode of binding of aminogly-cosides by RNA is currently a subject of intenseanalyses (Lato et al., 1995; Wallis et al., 1995; Wang& Rando, 1995; Famulok & HuÈ ttenhofer, 1996;Fourmy et al., 1996; Hendrix et al., 1997; Recht et al.,1996; Wang & Tor, 1997; Wang et al., 1997). Theself-splicing group I intron td from the T4 phagewas used as a model system for studying themode of action of neomycin B. Footprinting anal-ysis of neomycin B with the closely related sunYintron had previously revealed several bases in thecore of the intron which were protected fromchemical modi®cation in the presence of the anti-biotic (von Ahsen & Noller, 1993). We mutated theprotected bases and measured the effects of themutations on the td ribozyme's sensitivity towardsneomycin B. The in¯uence of Mg2� and pH onneomycin B inhibition as well as interactions withthe RNA backbone were analysed. Iodine cleavageof phosphorothioate-substituted RNA was used, inthe absence and presence of neomycin B, to detectchanges in the accessibility of the RNA backbonedue to antibiotic binding. A method using Fe2�-generated hydroxyl radicals was applied tomeasure the in¯uence of neomycin B on the bind-ing of metal ions to the intron core. Taken together,these data allowed docking of neomycin into the3D 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

which reveals structural principles for neomycinB/RNA interactions.

Results

Mutation of neomycin footprinting sites

Several bases in the group I intron core had pre-viously been shown to be protected from chemicalmodi®cation by neomycin B (von Ahsen & Noller,1993). To analyse the role of these bases in neomy-cin inhibition, we constructed a set of pointmutations in the td intron. Figure 1A shows themutations analysed in this study. Mutations atpositions C56 and G938 did not result in severesplicing de®ciency in vitro. At 10 mM MgCl2, themutant introns spliced with an activity similarto that of the wild-type (data not shown). TheG871C:C946G:G1016A triple mutant requires highconcentrations of ATP as the cofactor to splice ef®-ciently. This is in good agreement with the effect ofthe mutation in the Tetrahymena ribozyme (Been &Perrotta, 1991). A very sensitive assay for thymidy-late synthase activity and thus for ef®cient splicingof its intron is the in vivo phenotype on media lack-ing thymine (Belfort et al., 1987). Wild-type td�P6-2 and derived mutants were transformed into athymine-de®cient Escherichia coli strain and growthwas monitored on thymine-de®cient, trimethoprimand full media (Figure 1B). The mutants in J8/7and J5/4 grew well on the minimal medium andonly the C56G mutant showed an intermediatephenotype on trimethoprim-containing media,indicating that these mutations did not causemajor splicing defects. The other mutants used inthis work (C870U; C871C:C946G:G1016A andG871A:C946U (Michel et al., 1989) are splicingde®cient in vivo. Therefore, they cannot grow inthe absence of thymine while they are resistant totrimethoprim.

The mutations have only minor effects on theintron's sensitivity towards neomycin B

The in¯uence of Mg2� on neomycin inhibition ofthe wild-type was tested. At suboptimal Mg2� con-centrations (<3 mM), the intron is hypersensitive toneomycin B. However, between 5 and 10 mMMg2�, differences in neomycin sensitivity werenegligible and within the error range (Figure 2).Thus, all the in vitro splicing assays were per-formed at 10 mM Mg2� to take into account theincreased Mg2� requirement of several mutants. Toobtain comparable splicing ef®ciencies for mutantsand wild-type, GTP concentrations also had to beadjusted. As the required amounts of cofactor weresigni®cantly higher for some of the mutants, wetested the in¯uence of high GTP on neomycin inhi-bition of wild-type splicing. As shown in Table 1,the differences between the Ki values at 2.5 and100 mM GTP were insigni®cant. The rate limitingstep in splicing of the td intron is exon ligation(second step), which is monitored in these assays.

558 Splicing Inhibition by Neomycin B

Figure 1. A, Secondary structure of the T4 phage-derived td intron (adapted from Cech et al. (1994) withXRNA). Filled arrows indicate positions and nature ofmutations, open arrows indicate 50 and 30 splice sites (50ss and 30 ss). The G-binding site is surrounded by anopen square. P1 to P9.2 designate the stems, J8/7 is thesingle-stranded junction between P8 and P7. TheG871C:C946G:G1016A construct is a triple mutant.B, In vivo analysis-plating phenotypes. Constructs wereplated on selective media: thy�, non-selective medium;thyÿ, minimal medium lacking thymine; TTM, minimalmedium supplemented with thymine and trimethoprim.wt, wild-type; CGA, mutant G871C:C946G:G1016A;AU, mutant G871A:C946U; C870U, G938C, C56A,C56G, C56U, single mutants.

Splicing Inhibition by Neomycin B 559

This step is not dependent on guanosine. In allmutants, exon ligation was still the rate limitingstep.

The sensitivity of the mutant introns to neomy-cin B was compared with that of the wild-type.A representative splicing reaction of the mutant inJ8/7 (G938C) in the absence and presence of0.25 mM neomycin B is shown in Figure 3. Table 1indicates the Ki values for neomycin for the wild-type as well as for the mutants in J8/7, J5/4 andthe G-binding site. The mutations had only minoreffects on neomycin B sensitivity. These resultsclearly demonstrate that the mutations neither dis-rupt nor interfere with essential contacts betweenthe antibiotic and the intron RNA. Thus, we con-clude that the base protections observed previouslyresult from indirect contacts or from direct contactswith non-inhibitory neomycin molecule(s).

To probe changes in the accessibility of thebackbone due to antibiotic binding we used themethod developed by Eckstein and co-workers(Heidenreich et al., 1993; Schatz et al., 1991). Phos-phorothioates were randomly incorporated into the

RNA during in vitro transcription and the rena-tured RNA was subsequently cleaved with iodinein the absence and presence of neomycin B. Back-bone footprints of neomycin B accumulate in theP4/P6 domain with the joining regions J5/4 andJ6/7 being most prominently protected (data notshown and Figure 5B). Protections were onlydetected at antibiotic concentrations much higherthan the inhibitory concentrations. We must there-fore assume that the inhibitory neomycin mol-ecule(s) does not protect any phosphate or, due tosome inexplicable reason, it is only capable of pro-tecting the backbone from iodine at much higherconcentrations.

pH dependence of splicing inhibition byneomycin B

Several amino groups of neomycin B are proto-nated at pH 7 and deprotonated between pH 7and pH 9 (Figure 4A; Botto & Coxon, 1983). Forthe hammerhead ribozyme, the pH dependence ofinhibition suggested that at least three of the ®vepositively charged ammonium groups of neomycinB are critical for inhibition (Clouet-d'Orval et al.,1995). We measured inhibition of wild-type td spli-cing by neomycin B at different pH values, rangingfrom pH 7 to pH 8.5. Within this range, splicingactivity is not affected by pH changes (Herschlag& Khosla, 1994). As shown in Figure 4B, inhibitionis strongly pH dependent. At pH 7.5 inhibition isstill effective, whereas at pH 7.8 the ef®ciency ofneomycin is signi®cantly reduced and at pH 8

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

Table 1. Inhibition constants (Ki) of wild-type andmutants for neomycin B

Mutant GTP (mM) Ki (mM)

Wild-type 2.5 0.17 � 0.07Wild-type 100 0.11 � 0.05G938C 2.5 0.11 � 0.03C56A 50 0.48 � 0.24C56G 5 0.36 � 0.2C56U 25 0.26 � 0.08G871C : C946G : G1016A 1000a 0.36 � 0.14C870U 100 54 � 14

mM GTP � concentration of GTP at which Ki was deter-mined.

a The mutant G871C : C946G : G1016A requires ATP as acofactor for splicing instead of GTP.

Figure 2. Magnesium dependence of inhibition of thetd splicing reaction by neomycin B. v0/vi � ratio of spli-cing activity (exon ligation) in the absence and presenceof neomycin.


splicing is nearly unaffected by the antibiotic.Thus, protonated ammonium groups are mostprobably essential for binding of neomycin B to theintron RNA. The 20 and 2000 amino groups deproto-nate around pH 7.6. The importance of the 20amino group had been demonstrated earlier by astructure function analysis: kanamycin B, with a 20amino group inhibits splicing at 10 mM and kana-mycin A, with a 20 hydroxyl group as the onlydifference, does not inhibit at 5 mM (von Ahsenet al., 1991, 1992). The 2000 amino group is notexpected to be essential as ring IV is variable andnot conserved among aminoglycosides. The role ofthe 60 amino group cannot be determined by pHvariation, as the antibiotic loses its inhibitory

activity at pH 7.8, and the 60 amino group onlydeprotonates at pH 8.6. The importance of thisgroup is demonstrated by paromomycin, whichcontains, as the sole difference to neomycin B, anOH group at position 60, and kanamycin C, whichalso contains a hydroxyl group at this position.Paromomycin inhibits splicing at 36 mM (data notshown) and kanamycin C is inactive at 5 mM (vonAhsen et al., 1991).

Fe2�-generated hydroxyl radical cleavage inthe presence of neomycin B

To test whether divalent metal ion binding sitesin the group I intron are potential targets for theantibiotic, we employed a recently developedmethod to probe for the surroundings of metalions (Berens et al., 1998). Fe2�, which is similar toMg2� in both size and coordination geometry(Brown, 1988) can, in the absence of EDTA, struc-turally or functionally substitute for Mg2�, as hasbeen 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 sodiumascorbate and hydrogen peroxide, Fe2� generateshydroxyl radicals which can cleave the phospho-diester backbone (Latham & Cech, 1989; Tullius &Dombroski, 1985; Wang & Cech, 1992). Incubationof group I intron RNAs with Fe2� revealed severalconserved strong cleavage sites in the intron core(J5/4, J6/7, J8/7 and P7) as well as other cleavagesites in the peripheral extensions (Berens et al.,1998). To determine if these sites are affected byneomycin B, we cleaved the RNA with Fe2� in thepresence of increasing concentrations of this anti-biotic. As speci®city controls we used kanamycinA and spermidine. A typical cleavage gel is shownin Figure 5A. In the presence of neomycin B, sev-eral sites change in cleavage intensity. They arelocated in J5/4 (U72; not shown), in J6/7 (U867), inP7 (C870) and J8/7 (G938, U940, and U942). Sig-nals in L7.1 (G883, G884) and J7.2/3 (A907, A908)stem from a single peripheral metal ion bindingsite (Berens et al., 1998). In Figure 5B all protectionsare summarized. Of these, only A908 shows anincrease in cleavage intensity, all others are cleavedless ef®ciently. In addition, cleavage at four nucleo-tides is affected by both neomycin B and kanamy-cin A. G864 and A868 are cleaved more strongly,U889 and A890 less intensively. Protection fromFe2�-OH radical cleavage is detected at 5 mM neo-mycin, which is higher than the Ki of inhibition(0.17 mM). This is due to the assay conditionsnecessary for these experiments: the RNA concen-tration is 1 mM and thus the ratio RNA to antibioticis 1:5, which is also the ratio observed for inhi-bition. In the presence of spermidine (1 to 500 mM),these patterns do not change (data not shown),indicating that non-speci®c interactions with thebackbone do not affect the Fe2�-generated cleavagesites. Several sites affected by neomycin B are closeto the previously proposed A and B metal ionbinding sites (Streicher et al., 1996). The protection

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


of positions U72, U867 and C870 is a clear indi-cation that the A-site metal ion is displaced fromits original position. The same is true for the B-siteion 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 semicon-served and introns with C and A at this positionare active under physiological conditions. Mutationof this nucleotide in the td intron from C to U or Gresults in an increased requirement for Mg2� andfor the cofactor guanosine (Schroeder et al., 1991).Metal-induced cleavage of the RNA backboneresulted in the proposal that the N3 position of Cwas involved in the coordination of the essential

A-site metal ion (Streicher et al., 1996). Since thisposition was strongly protected from Fe2�-inducedcleavage in the presence of neomycin, we analysedthe sensitivity of the C870U variant towards neo-mycin B. Wild-type and the C870U constructs wereanalysed at identical conditions (10 mM MgCl2and 100 mM GTP) and their sensitivity towardsneomycin was compared. The Ki of the mutant forneomycin B was found to be 54 mM, while it is0.11 mM for the wild-type. Thus the C870U mutantis 500-fold more resistant to neomycin B than thewild-type (Table 1 and Figure 6A and B). The sen-sitivity of this mutant towards paromomycin wasalso tested and found to be 46 mM (data notshown), while the Ki for the wild-type is 36 mM.This indicates that the mutant cannot discriminatebetween neomycin and paromomycin, suggesting

Figure 5 (legend opposite)


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

Discussion

Chemical protection experiments with DMS andkethoxal, which probe for base contacts (von

Ahsen & Noller, 1993) and with iodine cleavage ofphosphorothioate-substituted RNA, which probesfor backbone contacts, revealed several footprintsin the presence of neomycin B scattered over the2D structure of the intron. Some of these positionsconverge to de®ned regions in space in the three-dimensional model available for the subgroup towhich the td intron belongs (Jaeger et al., 1993;Lehnert et al., 1996; Streicher et al., 1996). Twomajor sites of protection by neomycin B were

Figure 5. A, Probing for metal-binding sites in the td intron in the absence and presence of antibiotics. Intron RNAwas cleaved with Fe2�, sodium ascorbate and hydrogen peroxide after incubation with increasing amounts of neomy-cin B (Neo B) or kanamycin A (Kan A). G is a RNase T1 sequencing lane, AH an alkaline hydrolysis ladder. Severalsecondary structure elements of td are indicated on the left. On the right, nucleotide positions are indicated where theFe2�-mediated cleavage is strongly decreased in the presence of neomycin B, arrows indicate subtle effects. B, Second-ary 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 arecleaved less ef®ciently, open circles nucleotide positions which are cleaved more ef®ciently by Fe2�-generatedhydroxyl radicals in the presence of neomycin B (see A). Numbering of nucleotides and designation of secondarystructure elements as in Figure 1A.


detected, one in the internal loop between thestems P4 and P5 and the other one surroundingthe G-binding core. Mutational analyses in J4/5and J8/7 suggest that no interactions essential forinhibition of exon ligation occur between the pro-tected bases and the antibiotic. In contrast,mutation of a single position in the core (C870U)resulted in neomycin resistance, indicating thatbinding of neomycin B to this site is responsiblefor inhibition.

In previous work on the modelling of aminogly-cosides into the hammerhead ribozyme (Hermann& Westhof, 1998), molecular dynamics simulationsof neomycin B in aqueous and neutralized sol-utions were performed. In that report, it was

shown that the intramolecular distances betweenthe ammonium groups in the antibiotic matchedthe inter-ionic distances between the magnesiumions in the ribozyme as deduced from X-ray crys-tallography (Scott et al., 1996). Most importantly,the ammonium groups at positions 1 and 20 wereshown to replace the magnesium ions closest tothe cleavage site. We applied the same strategy tothe td intron and docked eight different conformersof neomycin B (extracted from the MD simulationsand representing the main conformational families)to the G-binding site so that the ions at sites A andB were displaced by ammonium groups on neomy-cin B rings I and II. Using this strategy, it wasnever possible to cover the two major protectedregions with one neomycin B molecule. We thusconcluded that there are at least two binding sitesfor neomycin B on the td intron.

One major site of protection lies in the internalloop between the stems P4 and P5 and was notmodelled precisely owing to the lack of data. Also,protection occurs at antibiotic concentrationswhich are much higher than for inhibition. Theasymmetric P4/P5 internal loop has been shown tobe involved in docking the P1 stem harbouring the50 splice site (Cech et al., 1994; Michel & Westhof,1990). There are several examples of asymmetricinternal loops which bind aminoglycoside anti-biotics: both the Rev responsive element of HIVand the decoding site of the 16 S rRNA are asym-metric internal loops, where the loops are closedby non-canonical base-pairs. In both cases the anti-biotic contacts the deep groove (Fourmy et al.,1996; Zapp et al., 1993). An in vitro selection forneomycin binding RNAs resulted in the isolationof stem-loops with helical irregularities, whichopen the deep groove (Wallis et al., 1995). Mostprobably, neomycin B sits on the deep groove sideof the P4/P5 loop on the opposite side of the P1recognition region. This loop should thus not bethe inhibitory binding site. The functional simi-larity between the P4/P5 domain of group Iintrons and the decoding site of 16 S rRNA A-siteis remarkable, as both RNAs bind a short RNAhelix, most probably via the 20 OH groups of theRNA backbone (Fourmy et al., 1996; Pyle et al.,1992; Schroeder et al., 1993; Strobel & Cech, 1994;Strobel et al., 1997). How neomycin binds to theinternal loop between P4 and P5 remains to beanalysed, but in analogy to the decoding process,aminoglycosides should not inhibit the docking ofthe 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 surroundsthe G-binding core and since a mutation at a singleposition (C870) in the core results in resistance, weconclude that this is the inhibitory site. The dock-ing of neomycin B to the G-binding core could beachieved by all conformers in such a way(Figure 7A, B and C) that (i) the two magnesium

Figure 6. Splicing inhibition of wild-type and mutantC870U by neomycin B. A, Wild-type precursor RNAwas renatured as described in Materials and Methodsand splicing was initiated by adding 100 mM GTP, andtime points up to 120 seconds were taken. Increase ofproduct (ligated exons) was plotted versus time and theplot was corrected for hydrolysis background. Slopesindicate % exon ligation per second. B, Mutant C870Uprecursor RNA was treated as the wild-type, timepoints were taken up to 150 seconds in the presence of0, 0.1 and 40 mM neomycin B, respectively. Formation ofligated exons was plotted as in A.


ions are displaced by the ammonium groups ofrings I and II, and (ii) the G-cofactor binding is nothindered (non-competitive inhibition). With allconformers tested, rings III and IV point towardsthe solvent where the substrate is expected to bind.The conformers distinguish themselves byrotations about the glycosyl bonds between rings Iand II. Thus, while ammonium 1 on ring II alwaysreplaced magnesium ion B, magnesium ion A wasalternatively replaced by the ammonium group 20or 60 on ring I. Thus, those aminoglycoside deriva-tives lacking an ammonium group at either 20 or 60are less effective inhibitors, since only a subset ofconformers present in solution can simultaneouslyreplace both magnesium ions.

Single point mutations have previously beenreported to be responsible for resistance to amino-glycosides. A single G to A mutation in the decod-ing site of the prokaryotic ribosome renderstranslation insensitive to aminoglycosides (DeStasio et al., 1989). Interestingly, an A to Gmutation in the human mitochondrial 12 S rRNAwas reported to create a high af®nity binding sitefor aminoglycosides, causing a hereditary form ofaminoglycoside-induced deafness (Hamasaki &Rando, 1997).

Our results clearly demonstrate that metal ionsare displaced by the antibiotic, as neomycin B pro-tects several distinct positions from cleavage. Sincedivalent metal ions are crucial for catalysis of mostribozymes, displacing them is a very ef®cient wayof inhibition of catalysis. For group I intron spli-cing, at least two metal ions are essential. Metalion-induced cleavage of the RNA backbone lead tothe structural model, where two metals surroundthe 50 splice site. These ions have been designatedA and B according to the Steitz & Steitz model(Steitz & Steitz, 1993; Streicher et al., 1996). Therequirement for both ions has been demonstratedby chemical interference studies (Piccirilli et al.,1993; Weinstein et al., 1997) and by kinetic exper-iments (McConnell et al., 1997). The metal ion Awas proposed to contact the bulged nucleotide inP7 (Streicher et al., 1996) and the G-cofactor(SjoÈgren et al., 1997). The metal ion B is in positionto coordinate the 30 bridging oxygen at the 50 splicesite (Piccirilli et al., 1993). Neomycin, as modelledinto the core of the td intron, displaces both ions;

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

carbon white. The yellow spheres represent the pro-posed positions for two Mg2� critical for catalysis(Streicher et al., 1996); ion A is close to the bulge C andion B at the bottom of the picture. Ring I of neomycin B(here nitrogen 20) is close to ion A and ring II (nitrogen1) to ion B. For clarity, metal ions are shown togetherwith neomycin B; in reality the metals are displaced inthe presence of the antibiotic. A, View down the G-bind-ing site. B, View after a rotation about a horizontal axisshowing rings III and IV above the G-cofactor. C, Space-®lling drawing of the complex between the td intronand neomycin B (in red, at the centre). In blue, domainP2; in yellow domain P9; and in orange domain P7. TheP4-P6 domain is at the back.


the 20 amino group of neomycin ring I displacesion A and the 1 amino group of ring II displacesion B (Figures 4A, 7A and B). Alternatively, sincerotation can occur around the glycosidic bondbetween rings I and II, the 60 amino group of ring Ican displace ion A. The modelling data are sup-ported by the resistance of the bulge mutant(C870U) to neomycin B and by the inability of thismutant to discriminate between neomycin B andparomomycin. The modelling data further suggestthat the N3 position of C870 is close to the 3 aminogroup of ring II, such that these two groupstogether with the 60 amino group form an equilat-eral triangle. However, we could not test this inter-action due to the lack of an aminoglycosideantibiotic with a 3 hydroxyl group in ring II.

Inhibition of ribozyme catalysis by neomycin Bmight in general occur by metal ion displacement.All ribozymes which have been tested to date forsensitivity towards aminoglycosides are indeedinhibited (Schroeder & von Ahsen, 1996). There isonly one ribozyme where no divalent metal ionappears to be required for the catalytic step: thehairpin ribozyme (Chowrira & Burke, 1991;Hampel & Cowan, 1997; Nesbit et al., 1997; Younget al., 1997). The very recent observation that thisribozyme is resistant to neomycin B (ZacharyTaylor & John Burke, personal communication) isin total agreement with our observation that metalion-binding sites are the speci®c targets of amino-glycosides in ribozymes.

Materials and Methods

Bacterial strains and plasmids

The E.coli strain used for in vivo tests in this study is athymine-de®cient derivative of C600 (Fÿ, supE44, thi-1,thr-1, leuB6, lacY1, tonA21, �thyA). In vitro splicingexperiments were performed with a truncated version ofthe 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 cleavageexperiments, 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 Casa-mino acids (Belfort et al., 1983) but lacking thymine (thyÿ

medium) was used to select for the Td� (TS�) pheno-type. TTM, a minimal medium supplemented with thy-mine (50 mg/ml) and trimethoprim (20 mg/ml), wasused to screen for the Tdÿ (TSÿ) phenotype, since onlycells lacking TS activity are able to grow on media con-taining the folate analogue trimethoprim. In addition, allmedia contained 100 mg/l ampicillin. Cells were grownat 37�C.

In vitro mutagenesis

For introduction of mutation C56X, a mixed oligo-nucleotide (50-TCTACTAGAGAGXTTCCCCGTTTAG-30,where X � A, T or C) was used. Deoxyuridine-substi-tuted single-stranded DNA was isolated following theprotocol of Kunkel et al. (1987). Annealing of the phos-phorylated oligonucleotide to single-stranded DNA,elongation and ligation were as described by Williamsonet al. (1989). After transformation, the colonies werescreened on TTM for the Tdÿ phenotype. MutantsG871A:C946U designated AU and G938C werekindly provided by T. Hirsch and mutantG871C:C946G:G1016A by Herbert Wank. The C870Uconstruct was reported previously (Schroeder et al.,1991).

RNA preparation

Precursor RNA (preRNA) of the T4 phage-derivedthymidylate synthase (td) intron was transcribed in vitrofrom plasmid td�P6-2 (Schroeder et al., 1991) with[a-35S]CTP and puri®ed according to the proceduredescribed by Streicher et al. (1993). Ribozyme constructtdL-7 was prepared by transcription under hydrolysisconditions (pH 8.0, 15 mM MgCl2). To obtain tdL-7 pre-cursor RNA for iodine backbone cleavage, transcriptionmixtures were doped with one of the phosphorothioateanalogues at a ratio of 10%. Transcriptions were per-formed under hydrolysis conditions, but otherwise asdescribed by Streicher et al. (1993).

In vitro splicing assays

For renaturation, the gel-puri®ed precursor RNA wasincubated with splicing buffer containing magnesium fortwo minutes at 56�C. Subsequently, a zero value wastaken, the reaction was started with GTP and time pointswere taken. Reactions were stopped by precipitationwith 0.3 M NaOAc/EtOH and reaction products wereseparated on 5% (w/v) denaturing acrylamide gels. Inthe case of mutant G871C:C946G:G1016A, to avoidhydrolysis, precursor RNA was not renatured after gelpuri®cation. For this mutant, the cofactor which initiatesthe reaction is ATP instead of GTP. The GTP require-ments of all other mutants were tested and GTP concen-trations were chosen in a way so as to obtain splicingactivities similar to that of the wild-type at 2.5 mM GTP.Unless otherwise indicated, all splicing and inhibitionexperiments were performed in a buffer containing50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 0.4 mMspermidine with 20 ng of precursor RNA. When the pHdependence of splicing inhibition was determined, incu-bation buffer was as described above but contained50 mM Hepes (pH 7.0, 7.2, 7.5, 8.0 or 8.5). PrecursorRNA was renatured as described above, antibiotic wasadded to the corresponding reactions and a zero valuewas taken. Subsequently, the reaction was started withGTP and incubated for two minutes at 37�C, while acontrol without GTP was incubated in the same way tocorrect for hydrolysis. Each experiment was done in tri-plicate. Gels were scanned on a PhosphorImager andanalysed as described below.

Ki determination

Splicing time courses of wild-type and mutants wereperformed as described above in the absence and pre-


sence of various neomycin B concentrations and gelswere scanned on a PhosphorImager. Increase per secondof ligated exons, expressed as percentage of the sum ofprecursor and all product bands, was monitored asinitial velocities in the absence (v0) and presence (vi) ofantibiotic. The values of v0/vi were plotted against anti-biotic concentrations, and Ki was determined as the con-centration at which v0/vi equals 2, which means that theinitial velocity is halved by the antibiotic.

Iodine cleavage of phosphorothioate substitutedprecursor RNA

Phosphorothioate substituted RNA was 50-end-labelled with [g-33P]ATP and preincubated with splicebuffer for three minutes at 56�C. Subsequently, antibioticwas added and allowed to bind for two minutes at roomtemperature. Iodine was added to a ®nal concentrationof 1 mM and the reaction was incubated at room tem-perature for one minute, stopped by the addition of fourvolumes stop solution (2.5 mM EDTA, 0.1 mg/ml yeasttRNA), precipitated with three volumes of 0.3 MNaOAc/EtOH and cleavage products were separated on6% denaturing acrylamide gels. Each experiment wasrepeated at least thrice, bands that turned out to bereproducibly protected were visually preselected andquanti®ed. The total radioactivity of each lane wasmeasured and the percentage of each band was deter-mined. By expressing the intensity of one band as thepercentage of the total radioactivity of a given lane,slight loading differences between lanes are neutralized.Protection was de®ned as signi®cant when the decreasewas at least 30%.

Fenton cleavage of antibiotic-treated RNA

50-[g-32P]- and 30-[a-32P]-labelling of RNA was asdescribed (Lingner & Keller, 1993; Sambrook et al., 1989).Sequencing ladders were generated by limited hydroly-sis with RNase T1 and NaHCO3 (Donis-Keller et al.,1977). For the Fe2�-mediated cleavage of antibiotic-trea-ted RNA, 1 ml tdL-7 RNA (6 pmol; approximately30,000 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 minutesat room temperature. 1 ml of either neomycin B, kanamy-cin A or spermidine (®nal concentrations ranging from0.5 to 500 mM) was added and the reaction incubated fortwo minutes at room temperature. 1 ml of 1.5 mM FeCl2was then added and incubated for one minute beforeadding 1 ml each of 15 mM sodium ascorbate and15 mM H2O2 to initiate the reaction. The reaction wasstopped after 60 seconds by adding thiourea to a ®nalconcentration of 150 mM. The RNA was precipitatedwith ethanol, dissolved in RNA-loading buffer andloaded onto 6% to 8% denaturing polyacrylamide gels.

Modelling

The aminoglycosides were manually docked into the3D model of the td intron using FRODO (Jones, 1978).The conformers used for the aminoglycosides werederived by molecular dynamics simulations using theprogram AMBER 4.1 (Pearlman et al., 1994) as describedby Herman & Westhof (1998). The 3D views were madeusing DRAWNA (Massire et al., 1994).

Acknowledgements

We are grateful to John Burke and Zachary Taylor forcommunicating results prior to publication. We thankHerbert Wank for the CGA mutant, Thomas Hirsch forthe G938C mutant, Norbert Polacek for the Ki of theCGA mutant, Thomas Hermann for the MD conformersof aminoglycosides, Bryn Weiser for the XRNA programand Norbert Polacek, Barbara Streicher and Mary G.Wallis for comments on the manuscript. E.W. thanks theInstitut Universitaire de France for support. This work isfunded by the Austrian Science Foundation (FWF) grantno. P11362 and P11999 to R.S. and by the EuropeanCommunity TMR program, grant no. FMRX-CT97-0154to 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)


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