ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2002, p. 1080–1085
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.4.1080–1085.2002
Copyright 2002, American Society for Microbiology. All Rights Reserved.
Oxazolidinone Antibiotics Target the P Site on
Hiroyuki Aoki,1 Lizhu Ke,1 Susan M. Poppe,2 Toni J. Poel,3 Elizabeth A. Weaver,3
Robert C. Gadwood,3 Richard C. Thomas,3 Dean L. Shinabarger,2 and M. Clelia Ganoza1*
Banting and Best Department of Medical Research, Toronto, Ontario M5G 1L6, Canada,1 and Infectious Diseases Research2 andMedicinal Chemistry,3 Pharmacia and Upjohn, Kalamazoo, Michigan 49001-0199
Received 30 April 2001/Returned for modification 20 September 2001/Accepted 18 January 2002
The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet- tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal 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.
A novel class of antimicrobial agents, the oxazolidinones,
onstrated that oxazolidinone footprints map to the central
target a wide spectrum of gram-positive and anaerobic bacteria
domain of the 16S rRNA whereas the 23S rRNA footprints
(4, 6, 9, 28). These compounds act by inhibiting protein syn-
map to domain V (20). Domain V is known to be involved in
thesis and have no effect on replication or transcription (8).
the peptidyl transferase reaction and in the binding of the 3Ј
Cell extracts exposed to oxazolidinones are impaired in protein
terminus of the tRNA substrates (23). However, efforts to
synthesis when programmed by native mRNAs but do not
demonstrate an effect of the oxazolidinones on the in vitro
appear to be inhibited when programmed by synthetic mRNAs
activity of the peptidyl transferase of ribosomes derived from
that lack the signals required for initiation and termination of
E. coli (this work) or from H. halobium (18) were unsuccessful.
translation (7, 8, 26). This suggested that these compounds
Here, we studied the effect of oxazolidinones on peptide
may target the initiation reaction. The oxazolidinone PNU-
bond formation in the presence of a protein, elongation factor
100766 (linezolid; Fig. 1) inhibits binding of the initiator fMet-
P (EF-P), that is essential for the optimum efficiency and
tRNA to the 70S ribosomal particle programmed with a syn-
specificity of peptide bond synthesis. We found that the oxazo-
thetic mRNA that harbors a Shine-Dalgarno sequence and a
lidinones inhibit EF-P-dependent peptide bond synthesis. To
properly spaced initiation codon (29).
examine this further, the fMet-tRNA was bound to the A site
Mutations to oxazolidinone resistance map to domain V of
and subsequently translocated to the P site with EF-G. The
the 23S rRNA in Halobacterium halobium (18), Staphylococcus
oxazolidinones were found to markedly inhibit the EF-G-me-
aureus (27), and the enterococci while mapping to domains IV
diated translocation of the fMet-tRNA from the ribosomal A
and V in Escherichia coli (33). The position of these PNU-
site to the P site. We propose that these antibiotics target the
100766 resistance mutations suggested to us that the oxazolidi-
ribosomal P site and inhibit initiation of protein synthesis by
nones may target peptidyl transferase indirectly by affecting
preventing the proper binding of fMet-tRNA.
the binding of the initiator tRNA. Since the P site accommo-
dates the initiator tRNA and the nascent protein, these drugs
could also affect the affinity of the peptidyl-tRNA for the
MATERIALS AND METHODS Bacteria and reagent preparation. [35S]methionine (1,175 Ci/mol) was pur-
Recent studies have indicated that the oxazolidinones bind
chased from ICN. Mid-log-phase strain MRE600 cells were purchased from the
to 70S ribosomes, as well as to 50S subunits (19), but not to 30S
University of Alabama Fermentation Facilities Center, Birmingham. The AUGtriplet was synthesized by using polynucleotide phosphorylase as previously de-
subunits. In contrast, a report by Matassova et al. (20) dem-
scribed (30). PNU-100766, PNU-140693, and PNU-176798 were obtained fromPharmacia Corp. Puromycin was labeled with 3H2O by incubation at 75°C for
12 h and then extracted into ethyl acetate. Assay of cell-free translation. Inhibition of cell-free translation was demon-
* Corresponding author. Mailing address: Banting and Best Depart-
strated by using the E. coli S30 Extract System for Circular DNA (Promega
ment of Medical Research, 112 College St., Toronto, Ontario M5G
Corp., Madison, Wis.). Each reaction mixture contained 2.5 l of S30 premix, 7.5
1L6, Canada. Phone: (416) 978-8918. Fax: (416) 978-8528. E-mail:
l of S30 extract, and 2.5 l of 10% drug–dimethyl sulfoxide (DMSO). Drugs
were dissolved in 30% DMSO and used in a final DMSO concentration of 5%
† M.C.G. dedicates this work to the memory of Clelia H. Finney.
during the assay. Control reaction mixtures received 5% DMSO and no drug.
OXAZOLIDINONES AFFECT THE RIBOSOMAL P SITE
TABLE 1. Antibacterial activity and translation inhibition
ultracentrifugation. The subunits were also stored in buffer A with 6 mM MgCl2
Initiation complex formation. Binding of the f[35S]Met-tRNA to E. coli 70S
ribosomes was conducted as previously described (8), except that initiation re-action mixtures were prepared without initiation factors and contained 6 mMmagnesium acetate [Mg(Ac)2], 0.08 M AUG, 30 mM NH4Cl, 10 mM Tris (pH
7.4), and 20 pmol of 70S ribosomes in a final volume of 60 l. The reactionmixtures were incubated for 15 min at 35°C, and the reactions were terminated
by addition of cold buffer A, washed with buffer A through Millipore filters, andcounted by liquid scintillation. Assay of peptidyl transferase and purification of EF-P. EF-P was purified as
previously described (1). EF-P-dependent peptidyl transferase activity was as-
sayed as described by Chung et al. (5) with the following modifications. Duringthe first step, the initiation complex was prepared with f[35S]Met-tRNA, AUG,and 70S ribosomes as described above for 20 min at 30C and then the sampleswere cooled to 0C. Unless otherwise specified, 1 M puromycin and 15%methanol were added in the second step and peptide bond formation was al-lowed to proceed for 5 min at 30C in the presence or absence of antibioticsand/or EF-P. PNU-176798 was first dissolved in 30% DMSO before addition tothe assay mixture. Control reaction mixtures contained DMSO instead of a drug.
FIG. 1. Structures of oxazolidinones PNU-100766 (I), PNU-140693
Peptidyl transferase fragment reaction. Peptidyl transferase was assayed in the
presence of ethanol and 60 mM MgC12 as described by Monro (21). Protein
concentration was 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
Reactions were initiated by the addition of 1 g (2.5 l) of plasmid pBestLuc and
min as described above, by using 2.5 mM MgCl2, 20 mM Tris (pH 7.5), 50 mM
incubated at 37°C for 30 min. Placement on ice for at least 5 min stopped the
NH4Cl, 0.10 mM dithiothreitol, and 0.1 mM GTP. Reaction mixtures were
reactions. A 1:8 dilution of the reaction mixture in dilution buffer was prepared,
subsequently incubated for 20 min at 4°C in the absence or presence of 1.5 g of
and 10 l was added to 50 l of luciferase reagent for reading of luminescence
EF-G. The EF-G recombinant protein was purified as previously described for
at 542 nm on a SpectraMAX Gemini (Molecular Devices). All reaction mixtures
the RbbA protein (16). The reactions were stopped by chilling on ice prior to the
were prepared in triplicate. An E. coli tolC::Tn 10 HN814 mutant from H.
addition of 0.4 M [3H]puromycin. Synthesis of f[35S]Met-[3H]puromycin was
Nikaido, University of California, Berkeley, was used to determine the effect of
measured by extraction of each reaction mixture with ethyl acetate, and the
the oxazolidinones in vivo. The tolC mutant is defective in the efflux pump that
radioactivity of the doubly labeled product was measured after addition of
prevents access of 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
suspended in 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 g/ml) was added, and the mixture
was incubated for 5 min. The suspension was centrifuged for 20 min at 13,000
In order to further examine the ability of oxazolidinones to
rpm (GSA rotor; Sorvall) to remove unbroken cells, debris, and alumina. The
inhibit the binding of fMet-tRNA to the ribosome, we used
centrifugation was repeated at 15,000 rpm for 30 min, and the supernatant was
three different compounds varying in potency against bacteria
centrifuged at 20,000 rpm for 18 h in a Ti 70 rotor (Beckman). The resulting
and cell-free translation (Fig. 1). Table 1 demonstrates that the
ribosome pellet was suspended by gentle stirring for 2 h in 2 to 3 ml of buf er A
potency of these oxazolidinones against bacteria correlated
containing 6 mM MgCl2. The suspension was then layered onto a 0 to 40%
sucrose gradient that was centrifuged for 18 h at 21,000 rpm in a swinging-bucket
well with their ability to inhibit cell-free translation, with PNU-
rotor (SW 40 Ti). One-milliliter fractions were collected from the top of the tube,
176798 proving to be approximately ninefold more potent than
and the A260 was monitored. The fractions containing the 70S ribosomes were
PNU-100766. Both compounds exhibited dose-dependent in-
combined and centrifuged at 24,000 rpm for 24 h in the Ti 70 rotor. The pellet
hibition of fMet-tRNA binding to 70S ribosomes; the 50%
was suspended in buffer A containing 6 mM MgCl2 and centrifuged again on a
second 0 to 40% sucrose gradient for 18 h at 18,000 rpm in a swinging-bucket
inhibitory concentrations (IC50s) of PNU-176798 and PNU-
rotor (SW 40 Ti). The fractions were identified by A
100766 for inhibition of 70S initiation complex formation were
above, and the 70S peak was isolated by ultracentrifugation for 24 h at 18,000
32 and 152 M, respectively (Table 1). Incubation of pre-
rpm in the Ti 70 rotor. The 70S ribosomes were suspended in buffer A containing
formed initiation complexes with 80 M PNU-176798 did not
6 mM MgCl2 and stored in small aliquots at Ϫ80°C.
result in destabilization (Fig. 2). Binding of fMet-tRNA was
Ribosomal subunits from the first centrifugation were isolated after dialysis of
the 70S ribosomes in buffer A containing 1 mM MgCl
inhibited by kanamycin, a well-established inhibitor of the ri-
isolated on sucrose gradients as described above and then concentrated by
bosomal P site (29). Conversely, fMet-tRNA binding was in-
FIG. 2. Effect of oxazolidinone PNU-176798 on the dissociation of
initiation complexes. The initiation complexes were formed as de-
scribed 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 M PNU-176798
(lanes 2, 4, and 6) or in its absence (lanes 1, 3, and 5). Reactions were
FIG. 4. Effect of oxazolidinone PNU-100766 on peptide bond for-
continued for 5 min at 35°C. The f[35S]Met-tRNA that remained
mation using the fragment reaction. The reactions were conducted at
bound to the ribosomes was then measured by filtration on nitrocel-
4°C as described by Monroe (21), and the reaction mixtures contained
lulose filters. Determinations were performed in duplicate.
60 mM MgCl2, 1 mM puromycin, 33% ethanol, and 50 pmol 70S or 50S
subunits. Determinations were performed in duplicate.
sensitive to the action of tetracycline, which impairs the ribo-
ple, in the presence of 66 M PNU-176798, the K
tRNA increased from about 0.1 M (no drug) to 0.8 M,
The kinetics of PNU-176798 inhibition were further exam-
indicating that PNU-176798 inhibition was competitive.
ined as a function of the fMet-tRNA concentration added to
Inhibition of fMet-tRNA binding results in inhibition of the
the initiation complex assay mixture. Figure 3 shows that the
peptidyl transferase reaction if the substrate is, indeed, bound
Km for fMet-tRNA increased with the antibiotic concentration,
to the ribosomal P site and is in proper juxtaposition with the
while the Vmax values remained relatively constant. For exam-
peptidyl transferase center of the 50S subunit. This reaction is
generally measured in the presence of organic solvents that
presumably help to bind the tRNA substrates to the ribosome,
as well as to activate the dormant peptidyl transferase. Under
such conditions, it was demonstrated that the peptidyl trans-
ferase of both the 50S subunit and 70S ribosomes is efficiently
inhibited by lincomycin and chloramphenicol, two well-known
inhibitors of peptidyl transferase (data not shown). However,
Fig. 4 shows that the reaction was impervious to the addition of
up to 2 mM PNU-100766 or the more potent compound PNU-
Several of the proteins known to be required for reconsti-
tution of synthesis (10, 12) were examined in attempts to in-
crease the efficiency of the peptidyl transferase reaction. As
shown in Fig. 5, addition of antibiotic to the reaction mixture
inhibits the activity of peptidyl transferase and results in dis-
cernible inhibition of peptidyl transferase by the oxazolidinone
PNU-176798, resulting in IC50s on the order of 40 M.
Figure 6 shows that in the presence of EF-P, more-pro-
nounced peptidyl transferase inhibition occurs when PNU-
176798 is added after formation of the initiation complex. Less
inhibition is observed when the antibiotic is added during bind-
FIG. 3. Lineweaver-Burke plots of the inhibition by oxazolidinone
ing of the substrate to the ribosome in the presence or absence
PNU-176798 of the fMet-puromycin formation. Formation of fMet-
puromycin was measured as described in Materials and Methods, by
To further examine whether the oxazolidinones preferen-
using different concentrations of f[35S]Met-tRNA. Each reaction mix-
ture contained the following concentrations of the antibiotic PNU-
tially affect the ribosomal P site, fMet-tRNA was bound to the
176798: F, no antibiotic; ■, 16.6 M; Œ, 50.0 M; , 66 M.
A site of the ribosome at 4C. Addition of EF-G and GTP
OXAZOLIDINONES AFFECT THE RIBOSOMAL P SITE
synthesis under the conditions described. Addition of PNU-
176798 markedly inhibited the EF-G-mediated translocation
of fMet-tRNA (Fig. 7B). Interestingly, examination of the ki-
netics of oxazolidinone inhibition of the translocation reaction
revealed that this translocation reaction is particularly sensitive
to inhibition, resulting in PNU-100766, PNU-140693, and
PNU-176798 IC50s of 110, 41, and 8 M, respectively (Table 1). DISCUSSION
The oxazolidinones selectively interfere with the protein syn-
thetic process (7, 8, 26), and PNU-100766 has been reported to
inhibit the formation of 70S initiation complexes in vitro (29).
The inhibition of initiation is consistent with a number of
biochemical experiments indicating that PNU-100766 does not
interfere with protein chain elongation directed by synthetic
random polymers or 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
FIG. 5. Effect of oxazolidinone PNU-176798 on EF-P-stimulated
effect on the forward initiation reaction, this oxazolidinone
synthesis of peptide bonds. Reactions were conducted with 15% meth-
exhibits no discernible effect on the stability of the initiation
anol, 1 M puromycin, and EF-P as described in Materials and Meth-
ods. The antibiotic was added in the first incubation during formation
reaction. The oxazolidinones acted as competitive inhibitors of
of the initiation complex. ᮀ, reaction mixtures without added EF-P.
Where indicated, 0.4 g of EF-P was added to each reaction mixture
Oxazolidinones have been reported to bind much more
strongly to 50S and 70S particles than to 30S subunits of ribo-
somes (19). Footprinting studies with a photoreactive oxazo-
lidinone demonstrated cross-linking to both the 16S and 23S
stimulated translocation of the initiator tRNA from the ribo-
rRNAs, binding to base A864 of the central domain of the 16S
somal A site to the P site, allowing peptide bond synthesis to
rRNA in a region that is not highly conserved (20). The foot-
occur in the presence of puromycin. The reaction was demon-
prints found on the 23S rRNA mapped to U2113, A2114,
strated to be dependent upon EF-G (Fig. 7A) and GTP (data
U2118, A2119, and C2153 (20), encompassing part of domain
not shown), resulting in a 40-fold increase in fMet-puromycin
V and extending into the region that binds ribosomal protein
L1 and comprise the tRNA exit (E) site (20). The oxazolidi-
none footprints that neighbor the E site may not define the
precise binding site, as cross-linking agents do not necessarily
target the exact site of ligand interaction. Alternatively, if the
antibiotics, indeed, bind to the E site, this binding could affect
the A site by a negative allosteric mechanism, as proposed for
protein chain elongation (24). The E site has also been clearly
demonstrated to influence the translocation reaction by affect-
ing the position of the 3Ј terminus of the P site-bound substrate
By using a mutagenized plasmid bearing the rrn operon and
either an acrAB or a tolC mutant, Xiong et al. mapped oxazo-
lidinone resistance in E. coli to base G2032 in a part of domain
IV of the 23S rRNA that interacts with domain V (33). All of
the other reported mutants map to several bases of domain V
of the 23S rRNAs of H. halobium, E. faecalis, and S. aureus (18,
27, 33). One simple interpretation of this finding is that the
oxazolidinones target the ribosomal P site in a region that has
several points of contact spanning both the 30S and 50S sub-
units. For the 50S subunit, the P site maps to G2252, A2451,
U2506, and U2585 (2), most of which are adjacent to the site
FIG. 6. Preferential effect of oxazolidinone PNU-176798 on the
of oxazolidinone resistance mutations (18, 27, 33). One of
initiation step preceding peptide bond synthesis. Initiation complex
these bases, A2451, has also been proposed to be the catalytic
formation was allowed to proceed for 20 min at 30°C with the indicated
residue of the peptidyl transferase (22).
concentrations of the antibiotic, and then 1 M puromycin and 0.4 g
The oxazolidinones cross-link to the 50S E site of domain V,
of EF-P were added (छ). The antibiotic was added after formation of
the initiation complex prior to the addition of puromycin and EF-P
and mutations to resistance involve several bases within the
ring structure of domain V that are part of the ribosomal P site
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: ᮀ, reaction mixtures containing EF-G; छ, reaction mixtures without EF-G.
(18, 33). Here we demonstrate that binding of fMet-tRNA to
EF-P accelerates peptide bond synthesis from aminoacyl-
ribosomes, EF-P-stimulated synthesis of peptide bonds, and
tRNAs or from CCA amino acids, which are poor acceptors of
the translocation reaction are impaired by the oxazolidinones.
ribosomal peptidyl transferase (10, 14). EF-P binds to the 30S
It is possible that these drugs bind with various affinities to
subunit and to 70S ribosomes and also interacts with the 50S
each ribosomal site or to a hybrid site of these particles. How-
particle. Discernible footprints can be detected in domain V of
ever, the simplest interpretation is that the oxazolidinones
the 50S subunit as a result of its interaction with EF-P (un-
principally target the P site of the ribosomes and subsequently
published data). Interestingly, EF-P protects U2555, A2564,
influence other ribosomal sites. The P site harbors the nascent
and C2576, which are near the site of eperezolid resistance in
chain that bears many amino acids that must be positioned
within the tunnel that spans both subunits. Thus, it is entirely
The crystal structure of EF-P from homologous protein
possible that the oxazolidinones also alter the P site in such a
eIF-5A of Methanococcus jannaschii indicates that the protein
fashion as to prevent entry of the peptide chain into the exit
has an elongated shape and that the N- and C-terminal ends of
tunnel. It has, in fact, been reported that oxazolidinones de-
the molecule are charge polarized (17). EF-P-stimulated syn-
crease the chain length of the nascent peptide (13), in keeping
thesis of peptide bonds is inhibited by streptomycin, which acts
with the idea that they interfere with their effect on the ribo-
on the 30S subunit, as well as lincomycin and chloramphenicol,
which impair the peptidyl transferase activity of the 50S sub-
If the P site of the ribosome is, indeed, affected by the
unit (1). These properties imply that EF-P could bind to both
oxazolidinones, these drugs would be expected to inhibit the
subunits as it activates peptide bond synthesis. This action of
peptidyl transferase when the tRNA substrate is properly po-
the protein might be brought about by its interactions with the
sitioned within the peptidyl transferase center. However, ef-
ribosome, which may then properly position the 3Ј terminus of
forts to detect peptidyl transferase inhibition by the oxazolidi-
the fMet-tRNA on the peptidyl transferase cavity prior to
nones have been unsuccessful in studies utilizing either E. coli
peptide bond synthesis. Alternatively, EF-P could enhance the
or H. halobium ribosomes (18, 27, 33). It is clear from both
affinity of the amino-acyl-tRNA (or the puromycin analogue)
previous reports and this work that the oxazolidinone IC50 for
the initiation reaction is significantly higher than that mea-
Study of the effect of the antibiotic during the initiation
sured for inhibition of cell-free translation (29). Therefore, in
reaction or after its completion, followed by addition of EF-P,
this study, we sought to enhance the sensitivity of the peptidyl
suggests that this oxazolidinone has an effect apart from inhi-
transferase assay by using the potent oxazolidinone
bition of initiation. Experiments reported here, in which the
PNU-176798 and EF-P. EF-P strongly stimulates peptide bond
fMet-tRNA was bound to the A site and then translocated to
synthesis (13, 14), and this synthesis was significantly inhibited
the P site by the action of EF-G (15), indicate that the antibi-
in this study by PNU-176798. EF-P has no direct effect on the
otic may, indeed, be capable of inhibiting translocation into the
binding of fMet-tRNA to the ribosome as measured by filtra-
ribosomal P site. Indeed, the IC50s of the oxazolidinone PNU-
tion assays (10). However, this protein could potentially help to
176798 are about 10-fold lower for translocation than for ini-
position fMet-tRNA in proper proximity to the peptidyl trans-
If the oxazolidinones target the ribosomal P site, one would
OXAZOLIDINONES AFFECT THE RIBOSOMAL P SITE
expect the synthesis of all polymers to be affected. However, it
9. Ford, C. W., J. C. Hamel, D. M. Wilson, J. K. Moerman, D. Stapert, R. J.
has been reported that synthesis directed by poly(U) (26) and
Yancey, D. K. Hutchinson, M. R. Barbachyn, and S. J. Brickner. 1996. In
vivo activities of U-100592 and U-100766, novel oxazolidinone antimicrobial
other synthetic polymers (7, 8) is impervious to the action of
agents, against experimental bacterial infections. Antimicrob. Agents Che-
these drugs. Thus, the oxazolidinones clearly inhibit translation
programmed by native templates, which harbor initiation sig-
10. Ganoza, M. C., and H. Aoki. 2000. Peptide bond synthesis: function of the efp
gene product. Biol. Chem. 381:553–559.
nals, but do not inhibit synthesis directed by random mRNAs
11. Ganoza, M. C., N. Barraclough, and J.-T. Wong. 1976. Purification and
properties of an N-formylmethionyl-tRNA hydrolase. Eur. J. Biochem. 65:
The translocation step from fMet-tRNA is preceded by a
12. Ganoza, M. C., C. Cunningham, and R. M. Green. 1985. Isolation and point
number of reactions that differ during synthesis of the first
of action of a factor from E. coli required to reconstruct translation. Proc.
peptide bond (25, 31). The initiator tRNA structural properties
Natl. Acad. Sci. USA 82:1648–1652.
13. Glick, B. R., and M. C. Ganoza. 1975. Identification of a soluble protein that
must be recognized in order to permit the joining of the sub-
stimulates peptide bond synthesis. Proc. Natl. Acad. Sci. USA 72:4257–4260.
units, as well as the approximation of the 3Ј end of fMet-tRNA
14. Glick, R. B., S. Chladek, and M. C. Ganoza. 1979. Peptide bond formation
to the 3Ј terminus of the incoming aminoacyl-tRNA, that must
stimulated by protein synthesis factor EF-P is dependent on the aminoacyl
moiety of the acceptor. Eur J. Biochem. 97:23–28.
occur prior to peptide bond synthesis. Unlike the elongation
15. Kevin, S., K. S. Wilson, and H. F. Noller. 1998. Mapping the position of
step that follows, the entrance of the second aminoacyl-tRNA
elongation factor EF-G in the ribosome by hydroxyl radical probing. Cell
into the ribosomes is not necessarily influenced by the filling of
16. Kiel, M. C., and M. C. Ganoza. 2000. Functional interactions of an Esche-
the E site with a tRNA that must exit the ribosome after
richia coli ribosomal ATPase. Eur. J. Biochem. 268:1–10.
peptide bond formation. Also, the first translocation may in-
17. Kim, K. K., H. Yokota, R. Kim, and S.-H. Kim. 1997. Cloning, expression
and crystallization of a hyperthermophilic protein that is homologous to the
volve dislodging of the mRNA-ribosome-fMet-tRNA complex
eukaryotic initiation factor, eIF-5A. Protein Sci. 6:2268–2270.
from the interaction of the 3Ј terminus of the 16S rRNA with
18. Kloss, P., L. Xiong, D. L. Shinabarger, A. S. Mankin. 1999. Resistance
the mRNA. The strong inhibition by the oxazolidinones of the
mutations in 23S rRNA identify the site of action of the protein synthesis
inhibitor linezolid in the ribosomal peptidyl transferase center. J. Mol. Biol.
translocation step could target one or more of these interme-
diate steps that underlie the special nature of the synthesis of
19. Lin, A. H., R. W. Murray, T. J. Vidmar, and K. R. Marotti. 1997. The
the first peptide bond. However, the simplest explanation for
oxazolidinone eperezolid binds to the 50S ribosomal subunit and competes
with binding of chloramphenicol and lincomycin. Antimicrob. Agents Che-
the mode of inhibition of the oxazolidinones is that they target
the ribosomal P site, thus exhibiting pleiotropic effects on sev-
20. Matassova, N. B., M. V. Rodnina, R. Endermann, H.-P. Kroll, V. Pleiss, H. Wild, and W. Wintermeyer. 1999. Ribosomal RNA is the target for oxazo-
eral intermediate steps of translation.
lidinones, a novel class of translational inhibitors. RNA 5:939–946.
21. Monroe, R. E. 1967. Catalysis of peptide bond formation by 50S ribosomal ACKNOWLEDGMENTS
subunits from Escherichia coli. J. Mol. Biol. 26:147–151.
22. Nissen, P., J. Hansen, N. Ban, P. B. Moore, and T. A. Steitz. 2000. The
We are grateful to K. Nierhaus for a generous gift of the overex-
structural basis of ribosome activity in peptide bond synthesis. Science 289:
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This work was supported by a grant from the Pharmacia Corp.
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Procedure Guideline for Diuretic Renographyin Children 3.0*Barry L. Shulkin1, Gerald A. Mandell2, Jeffrey A. Cooper3, Joe C. Leonard4, Massoud Majd5, Marguerite T. Parisi6,George N. Sfakianakis7, Helena R. Balon8, and Kevin J. Donohoe91St. Jude Children’s Research Hospital, Memphis, Tennessee; 2Phoenix Children’s Hospital, Phoenix, Arizona; 3Albany Medical Center,Albany, New York; 4Oklahoma
Double-Blind Placebo Controlled Crossover Study of ClotrimazoleWeek for Six Weeks in the Treatment of Rheumatoid ArthritisClotrimazole has a broad spectrum of anti-mycotic activity andwas developed clinically for its control of fungal infections,pathogenic for animals and man. The drug was developed as an agentby Bayer Laboratories from a screening program involving more than900 substituted