Oxazolidinone Antibiotics Target the P Site on Escherichiacoli Ribosomes

The oxazolidinones are a novel class of antimicrobial agentsthat target protein synthesis in a wide spectrum of gram-positiveand anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid)inhibits the binding of fMet-tRNA to 70S ribosomes. Mutationsto oxazolidinone resistance in Halobacterium halobium, Staphylococcusaureus, and Escherichia coli map at or near domain V of the23S rRNA, suggesting that the oxazolidinones may target thepeptidyl transferase region responsible for binding fMet-tRNA.This study demonstrates that the potency of oxazolidinones correspondsto increased inhibition of fMet-tRNA binding. The inhibitionof fMet-tRNA binding is competitive with respect to the fMet-tRNAconcentration, suggesting that the P site is affected. The fMet-tRNAreacts with puromycin to form peptide bonds in the presenceof elongation factor P (EF-P), which is needed for optimum specificityand efficiency of peptide bond synthesis. Oxazolidinone inhibitionof the P site was evaluated by first binding fMet-tRNA to theA site, followed by translocation to the P site with EF-G. Allthree of the oxazolidinones used in this study inhibited translocationof fMet-tRNA. We propose that the oxazolidinones target theribosomal P site and pleiotropically affect fMet-tRNA binding,EF-P stimulated synthesis of peptide bonds, and, most markedly,EF-G-mediated translocation of fMet-tRNA into the P site.

INTRODUCTION

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Introduction

A novel class of antimicrobial agents, the oxazolidinones, targeta wide spectrum of gram-positive and anaerobic bacteria (4,6, 9, 28). These compounds act by inhibiting protein synthesisand have no effect on replication or transcription (8). Cellextracts exposed to oxazolidinones are impaired in protein synthesiswhen programmed by native mRNAs but do not appear to be inhibitedwhen programmed by synthetic mRNAs that lack the signals requiredfor initiation and termination of translation (7, 8, 26). Thissuggested that these compounds may target the initiation reaction.The oxazolidinone PNU-100766 (linezolid; Fig. 1) inhibits bindingof the initiator fMet-tRNA to the 70S ribosomal particle programmedwith a synthetic mRNA that harbors a Shine-Dalgarno sequenceand a properly spaced initiation codon (29).

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FIG. 1. Structures of oxazolidinones PNU-100766 (I), PNU-140693 (II), and PNU-176798 (III).

Mutations to oxazolidinone resistance map to domain V of the23S rRNA in Halobacterium halobium (18), Staphylococcus aureus(27), and the enterococci while mapping to domains IV and Vin Escherichia coli (33). The position of these PNU-100766 resistancemutations suggested to us that the oxazolidinones may targetpeptidyl transferase indirectly by affecting the binding ofthe initiator tRNA. Since the P site accommodates the initiatortRNA and the nascent protein, these drugs could also affectthe affinity of the peptidyl-tRNA for the ribosome.

Recent studies have indicated that the oxazolidinones bind to70S ribosomes, as well as to 50S subunits (19), but not to 30Ssubunits. In contrast, a report by Matassova et al. (20) demonstratedthat oxazolidinone footprints map to the central domain of the16S rRNA whereas the 23S rRNA footprints map to domain V (20).Domain V is known to be involved in the peptidyl transferasereaction and in the binding of the 3' terminus of the tRNA substrates(23). However, efforts to demonstrate an effect of the oxazolidinoneson the in vitro activity of the peptidyl transferase of ribosomesderived from E. coli (this work) or from H. halobium (18) wereunsuccessful.

Here, we studied the effect of oxazolidinones on peptide bondformation in the presence of a protein, elongation factor P(EF-P), that is essential for the optimum efficiency and specificityof peptide bond synthesis. We found that the oxazolidinonesinhibit EF-P-dependent peptide bond synthesis. To examine thisfurther, the fMet-tRNA was bound to the A site and subsequentlytranslocated to the P site with EF-G. The oxazolidinones werefound to markedly inhibit the EF-G-mediated translocation ofthe fMet-tRNA from the ribosomal A site to the P site. We proposethat these antibiotics target the ribosomal P site and inhibitinitiation of protein synthesis by preventing the proper bindingof fMet-tRNA.

MATERIALS AND METHODS

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Materials and Methods

Bacteria and reagent preparation. [³⁵S]methionine (1,175 µCi/µmol) was purchased fromICN. Mid-log-phase strain MRE600 cells were purchased from theUniversity of Alabama Fermentation Facilities Center, Birmingham.The AUG triplet was synthesized by using polynucleotide phosphorylaseas previously described (30). PNU-100766, PNU-140693, and PNU-176798were obtained from Pharmacia Corp. Puromycin was labeled with³H₂O by incubation at 75°C for 12 h and then extracted intoethyl acetate.

Assay of cell-free translation. Inhibition of cell-free translation was demonstrated by usingthe E. coli S30 Extract System for Circular DNA (Promega Corp.,Madison, Wis.). Each reaction mixture contained 2.5 µlof S30 premix, 7.5 µl of S30 extract, and 2.5 µlof 10% drug-dimethyl sulfoxide (DMSO). Drugs were dissolvedin 30% DMSO and used in a final DMSO concentration of 5% duringthe assay. Control reaction mixtures received 5% DMSO and nodrug. Reactions were initiated by the addition of 1 µg(2.5 µl) of plasmid pBestLuc and incubated at 37°Cfor 30 min. Placement on ice for at least 5 min stopped thereactions. A 1:8 dilution of the reaction mixture in dilutionbuffer was prepared, and 10 µl was added to 50 µlof luciferase reagent for reading of luminescence at 542 nmon a SpectraMAX Gemini (Molecular Devices). All reaction mixtureswere prepared in triplicate. An E. coli tolC::Tn 10 HN814 mutantfrom H. Nikaido, University of California, Berkeley, was usedto determine the effect of the oxazolidinones in vivo. The tolCmutant is defective in the efflux pump that prevents accessof the drugs to the cell (27).

Preparation of ribosomes and ribosomal subunits. Ribosomes (70S) were isolated from mid-log-phase E. coli cells.The procedures were performed at 4°C. The cells (50-g lots)were broken by grinding with 100 g of alumina (Alcoa) and suspendedin 100 ml of buffer A (0.01 M Tris [pH 7.4], 0.001 M dithiothreitol,0.03 M NH₄Cl, 0.01 M MgCl₂). DNase (1.0 µg/ml) was added,and the mixture was incubated for 5 min. The suspension wascentrifuged for 20 min at 13,000 rpm (GSA rotor; Sorvall) toremove unbroken cells, debris, and alumina. The centrifugationwas repeated at 15,000 rpm for 30 min, and the supernatant wascentrifuged at 20,000 rpm for 18 h in a Ti 70 rotor (Beckman).The resulting ribosome pellet was suspended by gentle stirringfor 2 h in 2 to 3 ml of buffer A containing 6 mM MgCl₂. Thesuspension was then layered onto a 0 to 40% sucrose gradientthat was centrifuged for 18 h at 21,000 rpm in a swinging-bucketrotor (SW 40 Ti). One-milliliter fractions were collected fromthe top of the tube, and the A₂₆₀ was monitored. The fractionscontaining the 70S ribosomes were combined and centrifuged at24,000 rpm for 24 h in the Ti 70 rotor. The pellet was suspendedin buffer A containing 6 mM MgCl₂ and centrifuged again on asecond 0 to 40% sucrose gradient for 18 h at 18,000 rpm in aswinging-bucket rotor (SW 40 Ti). The fractions were identifiedby A₂₆₀ and collected as described above, and the 70S peak wasisolated by ultracentrifugation for 24 h at 18,000 rpm in theTi 70 rotor. The 70S ribosomes were suspended in buffer A containing6 mM MgCl₂ and stored in small aliquots at -80°C.

Ribosomal subunits from the first centrifugation were isolatedafter dialysis of the 70S ribosomes in buffer A containing 1mM MgCl₂. The subunits were isolated on sucrose gradients asdescribed above and then concentrated by ultracentrifugation.The subunits were also stored in buffer A with 6 mM MgCl₂ at-80°C.

Initiation complex formation. Binding of the f[³⁵S]Met-tRNA to E. coli 70S ribosomes was conductedas previously described (8), except that initiation reactionmixtures were prepared without initiation factors and contained6 mM magnesium acetate [Mg(Ac)₂], 0.08 µM AUG, 30 mM NH₄Cl,10 mM Tris (pH 7.4), and 20 pmol of 70S ribosomes in a finalvolume of 60 µl. The reaction mixtures were incubatedfor 15 min at 35°C, and the reactions were terminated byaddition of cold buffer A, washed with buffer A through Milliporefilters, and counted by liquid scintillation.

Assay of peptidyl transferase and purification of EF-P. EF-P was purified as previously described (1). EF-P-dependentpeptidyl transferase activity was assayed as described by Chunget al. (5) with the following modifications. During the firststep, the initiation complex was prepared with f[³⁵S]Met-tRNA,AUG, and 70S ribosomes as described above for 20 min at 30^oCand then the samples were cooled to 0^oC. Unless otherwise specified,1 µM puromycin and 15% methanol were added in the secondstep and peptide bond formation was allowed to proceed for 5min at 30^oC in the presence or absence of antibiotics and/orEF-P. PNU-176798 was first dissolved in 30% DMSO before additionto the assay mixture. Control reaction mixtures contained DMSOinstead of a drug.

Peptidyl transferase fragment reaction. Peptidyl transferase was assayed in the presence of ethanoland 60 mM MgC1₂ as described by Monro (21). Protein concentrationwas measured by the method of Bradford (3). The fMet-tRNA wasprepared as previously described (11).

EF-G-dependent translocation. Initiation complexes were formed at 4°C for 20 min as describedabove, by using 2.5 mM MgCl₂, 20 mM Tris (pH 7.5), 50 mM NH₄Cl,0.10 mM dithiothreitol, and 0.1 mM GTP. Reaction mixtures weresubsequently incubated for 20 min at 4°C in the absenceor presence of 1.5 µg of EF-G. The EF-G recombinant proteinwas purified as previously described for the RbbA protein (16).The reactions were stopped by chilling on ice prior to the additionof 0.4 µM [³H]puromycin. Synthesis of f[³⁵S]Met-[³H]puromycinwas measured by extraction of each reaction mixture with ethylacetate, and the radioactivity of the doubly labeled productwas measured after addition of scintillation fluid.

RESULTS

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Results

In order to further examine the ability of oxazolidinones toinhibit the binding of fMet-tRNA to the ribosome, we used threedifferent compounds varying in potency against bacteria andcell-free translation (Fig. 1). Table 1 demonstrates that thepotency of these oxazolidinones against bacteria correlatedwell with their ability to inhibit cell-free translation, withPNU-176798 proving to be approximately ninefold more potentthan PNU-100766. Both compounds exhibited dose-dependent inhibitionof fMet-tRNA binding to 70S ribosomes; the 50% inhibitory concentrations(IC₅₀s) of PNU-176798 and PNU-100766 for inhibition of 70S initiationcomplex formation were 32 and 152 µM, respectively (Table1). Incubation of preformed initiation complexes with 80 µMPNU-176798 did not result in destabilization (Fig. 2). Bindingof fMet-tRNA was inhibited by kanamycin, a well-establishedinhibitor of the ribosomal P site (29). Conversely, fMet-tRNAbinding was insensitive to the action of tetracycline, whichimpairs the ribosomal A site (data not shown).

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TABLE 1. Antibacterial activity and translation inhibition by oxazolidinones

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FIG. 2. Effect of oxazolidinone PNU-176798 on the dissociation of initiation complexes. The initiation complexes were formed as described in Materials and Methods and were undiluted (lanes 1 and 2) or diluted 10-fold (lanes 3 and 4) or 20-fold (lanes 5 and 6) in buffer A containing 6 mM Mg(Ac)₂ in the presence of 80 µM PNU-176798 (lanes 2, 4, and 6) or in its absence (lanes 1, 3, and 5). Reactions were continued for 5 min at 35°C. The f[³⁵S]Met-tRNA that remained bound to the ribosomes was then measured by filtration on nitrocellulose filters. Determinations were performed in duplicate.

The kinetics of PNU-176798 inhibition were further examinedas a function of the fMet-tRNA concentration added to the initiationcomplex assay mixture. Figure 3 shows that the K_m for fMet-tRNAincreased with the antibiotic concentration, while the V_maxvalues remained relatively constant. For example, in the presenceof 66 µM PNU-176798, the K_m for fMet-tRNA increased fromabout 0.1 µM (no drug) to 0.8 µM, indicating thatPNU-176798 inhibition was competitive.

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FIG. 3. Lineweaver-Burke plots of the inhibition by oxazolidinone PNU-176798 of the fMet-puromycin formation. Formation of fMet-puromycin was measured as described in Materials and Methods, by using different concentrations of f[³⁵S]Met-tRNA. Each reaction mixture contained the following concentrations of the antibiotic PNU-176798: •, no antibiotic; ${blacksquare}$ , 16.6 µM; ${blacktriangleup}$ , 50.0 µM; ${blacktriangledown}$ , 66 µM.

Inhibition of fMet-tRNA binding results in inhibition of thepeptidyl transferase reaction if the substrate is, indeed, boundto the ribosomal P site and is in proper juxtaposition withthe peptidyl transferase center of the 50S subunit. This reactionis generally measured in the presence of organic solvents thatpresumably help to bind the tRNA substrates to the ribosome,as well as to activate the dormant peptidyl transferase. Undersuch conditions, it was demonstrated that the peptidyl transferaseof both the 50S subunit and 70S ribosomes is efficiently inhibitedby lincomycin and chloramphenicol, two well-known inhibitorsof peptidyl transferase (data not shown). However, Fig. 4 showsthat the reaction was impervious to the addition of up to 2mM PNU-100766 or the more potent compound PNU-176798 (data notshown).

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FIG. 4. Effect of oxazolidinone PNU-100766 on peptide bond formation using the fragment reaction. The reactions were conducted at 4°C as described by Monroe (21), and the reaction mixtures contained 60 mM MgCl₂, 1 mM puromycin, 33% ethanol, and 50 pmol 70S or 50S subunits. Determinations were performed in duplicate.

Several of the proteins known to be required for reconstitutionof synthesis (10, 12) were examined in attempts to increasethe efficiency of the peptidyl transferase reaction. As shownin Fig. 5, addition of antibiotic to the reaction mixture inhibitsthe activity of peptidyl transferase and results in discernibleinhibition of peptidyl transferase by the oxazolidinone PNU-176798,resulting in IC₅₀s on the order of 40 µM.

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FIG. 5. Effect of oxazolidinone PNU-176798 on EF-P-stimulated synthesis of peptide bonds. Reactions were conducted with 15% methanol, 1 µM puromycin, and EF-P as described in Materials and Methods. The antibiotic was added in the first incubation during formation of the initiation complex. ${square}$ , reaction mixtures without added EF-P. Where indicated, 0.4 µg of EF-P was added to each reaction mixture ( ${blacksquare}$ ).

Figure 6 shows that in the presence of EF-P, more-pronouncedpeptidyl transferase inhibition occurs when PNU-176798 is addedafter formation of the initiation complex. Less inhibition isobserved when the antibiotic is added during binding of thesubstrate to the ribosome in the presence or absence of EF-P.

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FIG. 6. Preferential effect of oxazolidinone PNU-176798 on the initiation step preceding peptide bond synthesis. Initiation complex formation was allowed to proceed for 20 min at 30°C with the indicated concentrations of the antibiotic, and then 1 µM puromycin and 0.4 µg of EF-P were added ( ${diamond}$ ). The antibiotic was added after formation of the initiation complex prior to the addition of puromycin and EF-P ( ${square}$ ).

To further examine whether the oxazolidinones preferentiallyaffect the ribosomal P site, fMet-tRNA was bound to the A siteof the ribosome at 4^oC. Addition of EF-G and GTP stimulatedtranslocation of the initiator tRNA from the ribosomal A siteto the P site, allowing peptide bond synthesis to occur in thepresence of puromycin. The reaction was demonstrated to be dependentupon EF-G (Fig. 7A) and GTP (data not shown), resulting in a40-fold increase in fMet-puromycin synthesis under the conditionsdescribed. Addition of PNU-176798 markedly inhibited the EF-G-mediatedtranslocation of fMet-tRNA (Fig. 7B). Interestingly, examinationof the kinetics of oxazolidinone inhibition of the translocationreaction revealed that this translocation reaction is particularlysensitive to inhibition, resulting in PNU-100766, PNU-140693,and PNU-176798 IC₅₀s of 110, 41, and 8 µM, respectively(Table 1).

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FIG. 7. Effect of EF-G on the translocation of fMet-tRNA from the ribosomal A site to the P site. Initiation complexes were formed as described in Materials and Methods. The reactions were performed at 0°C and, where indicated, contained 1.5 pmol of EF-G and/or the designated concentrations of the oxazolidinone PNU-176798. (A) Reactions in the presence (column 1) or absence (column 2) of EF-G. (B) Oxazolidinone concentration required to inhibit EF-G-dependent translocation. The f[³⁵S]Met-puromycin product was measured by ethyl acetate extraction as described in Materials and Methods. Symbols: ${square}$ , reaction mixtures containing EF-G; ${diamond}$ , reaction mixtures without EF-G.

DISCUSSION

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Discussion

The oxazolidinones selectively interfere with the protein syntheticprocess (7, 8, 26), and PNU-100766 has been reported to inhibitthe formation of 70S initiation complexes in vitro (29). Theinhibition of initiation is consistent with a number of biochemicalexperiments indicating that PNU-100766 does not interfere withprotein chain elongation directed by synthetic random polymersor with the codon-dependent termination reaction (19, 26). Here,we report that the initiation reaction is also impaired by PNU-176798,a compound that is a potent inhibitor of translation in vivo.In contrast to its inhibitory effect on the forward initiationreaction, this oxazolidinone exhibits no discernible effecton the stability of the initiation reaction. The oxazolidinonesacted as competitive inhibitors of fMet-tRNA binding to theribosome.

Oxazolidinones have been reported to bind much more stronglyto 50S and 70S particles than to 30S subunits of ribosomes (19).Footprinting studies with a photoreactive oxazolidinone demonstratedcross-linking to both the 16S and 23S rRNAs, binding to baseA864 of the central domain of the 16S rRNA in a region thatis not highly conserved (20). The footprints found on the 23SrRNA mapped to U2113, A2114, U2118, A2119, and C2153 (20), encompassingpart of domain V and extending into the region that binds ribosomalprotein L1 and comprise the tRNA exit (E) site (20). The oxazolidinonefootprints that neighbor the E site may not define the precisebinding site, as cross-linking agents do not necessarily targetthe exact site of ligand interaction. Alternatively, if theantibiotics, indeed, bind to the E site, this binding couldaffect the A site by a negative allosteric mechanism, as proposedfor protein chain elongation (24). The E site has also beenclearly demonstrated to influence the translocation reactionby affecting the position of the 3' terminus of the P site-boundsubstrate (32).

By using a mutagenized plasmid bearing the rrn operon and eitheran acrAB or a tolC mutant, Xiong et al. mapped oxazolidinoneresistance in E. coli to base G2032 in a part of domain IV ofthe 23S rRNA that interacts with domain V (33). All of the otherreported mutants map to several bases of domain V of the 23SrRNAs of H. halobium, E. faecalis, and S. aureus (18, 27, 33).One simple interpretation of this finding is that the oxazolidinonestarget the ribosomal P site in a region that has several pointsof contact spanning both the 30S and 50S subunits. For the 50Ssubunit, the P site maps to G2252, A2451, U2506, and U2585 (2),most of which are adjacent to the site of oxazolidinone resistancemutations (18, 27, 33). One of these bases, A2451, has alsobeen proposed to be the catalytic residue of the peptidyl transferase(22).

The oxazolidinones cross-link to the 50S E site of domain V,and mutations to resistance involve several bases within thering structure of domain V that are part of the ribosomal Psite (18, 33). Here we demonstrate that binding of fMet-tRNAto ribosomes, EF-P-stimulated synthesis of peptide bonds, andthe translocation reaction are impaired by the oxazolidinones.It is possible that these drugs bind with various affinitiesto each ribosomal site or to a hybrid site of these particles.However, the simplest interpretation is that the oxazolidinonesprincipally target the P site of the ribosomes and subsequentlyinfluence other ribosomal sites. The P site harbors the nascentchain that bears many amino acids that must be positioned withinthe tunnel that spans both subunits. Thus, it is entirely possiblethat the oxazolidinones also alter the P site in such a fashionas to prevent entry of the peptide chain into the exit tunnel.It has, in fact, been reported that oxazolidinones decreasethe chain length of the nascent peptide (13), in keeping withthe idea that they interfere with their effect on the ribosomalP site.

If the P site of the ribosome is, indeed, affected by the oxazolidinones,these drugs would be expected to inhibit the peptidyl transferasewhen the tRNA substrate is properly positioned within the peptidyltransferase center. However, efforts to detect peptidyl transferaseinhibition by the oxazolidinones have been unsuccessful in studiesutilizing either E. coli or H. halobium ribosomes (18, 27, 33).It is clear from both previous reports and this work that theoxazolidinone IC₅₀ for the initiation reaction is significantlyhigher than that measured for inhibition of cell-free translation(29). Therefore, in this study, we sought to enhance the sensitivityof the peptidyl transferase assay by using the potent oxazolidinonePNU-176798 and EF-P. EF-P strongly stimulates peptide bond synthesis(13, 14), and this synthesis was significantly inhibited inthis study by PNU-176798. EF-P has no direct effect on the bindingof fMet-tRNA to the ribosome as measured by filtration assays(10). However, this protein could potentially help to positionfMet-tRNA in proper proximity to the peptidyl transferase center.

EF-P accelerates peptide bond synthesis from aminoacyl-tRNAsor from CCA amino acids, which are poor acceptors of ribosomalpeptidyl transferase (10, 14). EF-P binds to the 30S subunitand to 70S ribosomes and also interacts with the 50S particle.Discernible footprints can be detected in domain V of the 50Ssubunit as a result of its interaction with EF-P (unpublisheddata). Interestingly, EF-P protects U2555, A2564, and C2576,which are near the site of eperezolid resistance in S. aureus(27).

The crystal structure of EF-P from homologous protein eIF-5Aof Methanococcus jannaschii indicates that the protein has anelongated shape and that the N- and C-terminal ends of the moleculeare charge polarized (17). EF-P-stimulated synthesis of peptidebonds is inhibited by streptomycin, which acts on the 30S subunit,as well as lincomycin and chloramphenicol, which impair thepeptidyl transferase activity of the 50S subunit (1). Theseproperties imply that EF-P could bind to both subunits as itactivates peptide bond synthesis. This action of the proteinmight be brought about by its interactions with the ribosome,which may then properly position the 3' terminus of the fMet-tRNAon the peptidyl transferase cavity prior to peptide bond synthesis.Alternatively, EF-P could enhance the affinity of the amino-acyl-tRNA(or the puromycin analogue) by binding to the ribosome.

Study of the effect of the antibiotic during the initiationreaction or after its completion, followed by addition of EF-P,suggests that this oxazolidinone has an effect apart from inhibitionof initiation. Experiments reported here, in which the fMet-tRNAwas bound to the A site and then translocated to the P siteby the action of EF-G (15), indicate that the antibiotic may,indeed, be capable of inhibiting translocation into the ribosomalP site. Indeed, the IC₅₀s of the oxazolidinone PNU-176798 areabout 10-fold lower for translocation than for initiation (Table1).

If the oxazolidinones target the ribosomal P site, one wouldexpect the synthesis of all polymers to be affected. However,it has been reported that synthesis directed by poly(U) (26)and other synthetic polymers (7, 8) is impervious to the actionof these drugs. Thus, the oxazolidinones clearly inhibit translationprogrammed by native templates, which harbor initiation signals,but do not inhibit synthesis directed by random mRNAs lackingthese signals.

The translocation step from fMet-tRNA is preceded by a numberof reactions that differ during synthesis of the first peptidebond (25, 31). The initiator tRNA structural properties mustbe recognized in order to permit the joining of the subunits,as well as the approximation of the 3' end of fMet-tRNA to the3' terminus of the incoming aminoacyl-tRNA, that must occurprior to peptide bond synthesis. Unlike the elongation stepthat follows, the entrance of the second aminoacyl-tRNA intothe ribosomes is not necessarily influenced by the filling ofthe E site with a tRNA that must exit the ribosome after peptidebond formation. Also, the first translocation may involve dislodgingof the mRNA-ribosome-fMet-tRNA complex from the interactionof the 3' terminus of the 16S rRNA with the mRNA. The stronginhibition by the oxazolidinones of the translocation step couldtarget one or more of these intermediate steps that underliethe special nature of the synthesis of the first peptide bond.However, the simplest explanation for the mode of inhibitionof the oxazolidinones is that they target the ribosomal P site,thus exhibiting pleiotropic effects on several intermediatesteps of translation.

ACKNOWLEDGMENTS

We are grateful to K. Nierhaus for a generous gift of the overexpressingplasmid harboring the fusA gene encoding EF-G.

This work was supported by a grant from the Pharmacia Corp.

FOOTNOTES

* Corresponding author. Mailing address: Banting and Best Department of Medical Research, 112 College St., Toronto, Ontario M5G 1L6, Canada. Phone: (416) 978-8918. Fax: (416) 978-8528. E-mail: m.ganoza{at}utoronto.ca.

M.C.G. dedicates this work to the memory of Clelia H. Finney.

REFERENCES

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