EF-P also protects domain V of the 23S rRNA proximal to the A-site 50S T-site and more strongly the A-site of 70S ribosomes.. Thus, the action of EF-P may be similar to that of initiatio
Trang 1coli ribosome
Hiroyuki Aoki1, John Xu1, Andrew Emili2, John G Chosay3, Ashkan Golshani4and
M Clelia Ganoza1
1 University of Toronto, C.H Best Institute, Canada
2 Terrance Donnelly Centre for Cellular and Biomolecular Research (Donnelly CCBR), Toronto, Canada
3 Pfizer Pharmaceuticals, Ann Arbor, MI, USA
4 Department of Biology, Carleton University, Ottawa, Canada
In all cells, ribosomes are inert in the absence of
several proteins that promote each facet of protein
synthesis In eubacteria, initiation factors (IF-1, IF-2,
IF-3) bind to the 30S subunit, whereas elongation
factors (EF-Tu and EF-G) and termination factors
(RF-1, RF-2 and RF-3) bind to 70S ribosomes
Reconstitution of synthesis using all of these
homoge-neous proteins revealed that they are insufficient for
synthesis directed by a native mRNA and that several
other proteins are required [1–6] One of these
pro-teins, EF-P, stimulates peptide bond synthesis by
70S ribosomes [1,2,6–8] The gene encoding EF-P,
efp, occurs at a unique site at 94.3 min on the
Esc-herichia coli chromosome [9] Interruption of the efp
gene is lethal to the cell and results in an abrupt
cessation of protein synthesis, specifically in an
impairment of peptide-bond formation [10] Cells
deleted for efp grow only in the presence of the efp gene in trans Thus, the efp gene is essential for cell growth and viability [10,11]
Reconstitution experiments and the effect of various antibiotics suggest that EF-P binds to a site on ribo-somes that differs from the binding site of most other translation factors Thus, EF-P requires ribosomal pro-tein L16 for its action, but not L7⁄ L12, L6 or L11 [12] All of the G proteins, IF-2, EF-Tu, EF-G and RF-3 act with L7⁄ L12 in the sarcin-ricin loop to trig-ger GTP hydrolysis, and L6 is required for the action
of the release factors [6] Several antibiotics perturb the action of EF-P Notably, streptomycin, which impairs translational fidelity, is a strong inhibitor of the EF-P reaction [12] However, other antibiotics that alter the fidelity of decoding such as neomycin and kasugamycin, have no effect on the EF-P-mediated
Keywords
A-site; eIF5A; elongation factor EF-P;
ribosomes; translocation
Correspondence
H Aoki, University of Toronto, C.H Best
Institute, 112 College Street, Toronto,
Ontario M5G 1L6, Canada
Fax: +1 416 978 8528
Tel: +1 416 978 8918
E-mail: hiro.dr.aoki@utoronto.ca
(Received 1 August 2007, revised 3
Decem-ber 2007, accepted 10 DecemDecem-ber 2007)
doi:10.1111/j.1742-4658.2007.06228.x
EF-P (eubacterial elongation factor P) is a highly conserved protein essen-tial for protein synthesis We report that EF-P protects 16S rRNA near the G526 streptomycin and the S12 and mRNA binding sites (30S T-site) EF-P also protects domain V of the 23S rRNA proximal to the A-site (50S T-site) and more strongly the A-site of 70S ribosomes We suggest that EF-P: (a) may play a role in translational fidelity and (b) prevents entry of fMet–tRNA into the A-site enabling it to bind to the 50S P-site
We also report that EF-P promotes a ribosome-dependent accommodation
of fMet–tRNA into the 70S P-site
Abbreviations
CMCT, 1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide metho-p-toluenesulfonate; DMS, dimethyl sulfate; EF-P, eubacterial elongation factor P; IF, initiation factor; Ke, kethoxal; RF, termination factor.
Trang 2synthesis of peptide bonds [12] Also, translocation
inhibitors have little or no affect on the EF-P reaction
[12] EF-P enhances the inhibition of peptide-bond
for-mation by chloramphenicol and lincomycin [12] These
results imply that EF-P acts near the
peptidyltransfer-ase center of the ribosome
EF-P is required for synthesis with native mRNA
templates and for synthesis of poly(Phe) primed by
N-acetyl-Phe-tRNA as the initiator [1,2,12] The EF-P
protein is essential for the synthesis of certain
fMet-ini-tiated dipeptides [8,12] Thus, the action of EF-P may
be similar to that of initiation factor eIF-5A in
eukary-otes, which preferentially promotes synthesis of the
first peptide bond in the protein sequence [13–15] This
is further substantiated by the fact that EF-P harbors
84 and 64% sequence similarity with aIF5-A from
archaebacteria and eIF-5A from eukaryotes,
respect-ively [16–18]
Remarkably, the crystal structure of EF-P is an L,
or tRNA-like shape, characteristic of many proteins
that actuate translation [16–18]
The function of EF-P and eIF-5A are currently
unknown It has been proposed that these proteins
stimulate the synthesis of certain proteins in the cell
[19] In mammalian cells, including in humans, the
genes encoding eIF-5A and its isoform, eIF-5A2, are
oncogenes [19] Despite these observations, the specific
mode of action of these proteins in translation remains
enigmatic
Here, we examine interactions between EF-P and
70S ribosomes using several rRNA structure-specific
chemical probes [20,21] The nature of the interactions
between specific rRNA domains and EF-P suggests a
plausible mode of action for this ubiquitous protein
Results
Three approaches were undertaken to study the
posi-tion of the EF-P protein on the Escherichia coli
70S ribosome and its subunits
Binding of labeled EF-P to ribosomes
To study the binding site of EF-P on ribosomes, we
first ascertained that ribosomes were free of EF-P This
was accomplished by washing the ribosomes in 0.5 m
NH4C1 buffer and isolating the 70S peak after two
successive sucrose-density gradient centrifugation steps
as described in Experimental procedures [22] Western
blotting of the purified 70S preparations revealed that
the ribosomes were free of EF-P (data not shown) A
specific anti-EF-P mouse mAb was used to detect the
protein
The purity of the EF-P was first assessed by isoelec-tric focusing in the first dimension followed by SDS electrophoresis as described previously [23] and by subsequent immunoblotting with specific anti-(EF-P) mAbs A single band was observed on Coomasie Bril-liant Blue-stained gels and on the corresponding im-munoblots of the EF-P protein (Fig 1A) Thus, the EF-P protein appears to be homogeneous by these cri-teria This was verified by MS analysis of the tryptic fragments of the protein (see Experimental proce-dures) From the MS analysis of EF-P it was found that the residue blocking Lys34 conforms to spermi-dine within ± 1 Da (Table 1) No other known com-pound was found to more closely fit this mass
The N-terminus of the purified EF-P protein was labeled by formylation with [14C]formate and the protein was bound to partly dissociated 70S ribo-somes containing 30S and 50S subunits, as described
in Experimental procedures The ribosomeÆ[14C]EF-P complex was sedimented on sucrose-density gradients and the radioactivity of the different fractions was mea-sured EF-P was found associated with 70S ribosomes and 30S and 50S subunits Table 2 shows the amount
of EF-P that binds to each subunit and to 70S ribo-somes The labeled protein binds in an 1 : 1 molar ratio to 70S ribosomes and to 30S and 50S subunits
Identification of EF-P by immunoprecipitation
of ribosomes and their subunits
To examine whether EF-P occurs bound to native polyribosomes, a mouse mAb specific to EF-P was used to detect the protein As shown in Fig 1B, the EF-P protein can be detected on 70S, 30S and 50S particles and on polyribosomes Fractions from the sucrose-density gradients were collected and sub-jected to isoelectric focusing in the first dimension followed by 1D SDS electrophoresis and western blotting As shown in Fig 1B, EF-P is bound to the 30S and 50S subunits, to 70S ribosomes and to poly-ribosomes The density of the immunoblots was determined, as was the total area of each peak in the ribosome profile The percentage of bound EF-P protein in each peak shows that EF-P is distributed equally between 30S and 50S subunits and on 70S ribosomes Approximately 17% of the protein was found on 70S ribosomes and 7% occurred bound to the 30S and 50S subunits, respectively The reminder of the protein was recovered in the polyribosome fraction The bound EF-P fraction decreases as a function of the number of ribosomes
in the polyribosome fractions (Fig 1C) This suggests that EF-P acts in an initial stage of synthesis
Trang 3EF-P binding site on the ribosome
To further examine the interactions of EF-P,
ribo-somes were treated with various base-specific reagents
that react with unpaired bases at the ring nitrogen of each exposed rRNA base The sequence of the probes used spanned the 16S and 23S rRNA regions that are exposed to each reagent [20] We first used dimethyl
A
Fig 1 (A) Immunoelectrophoretic analysis
of purified EF-P Purified EF-P protein was
visualized with Coomasie Brilliant Blue and
detected on immunoblots of the
electropho-retic fractions using a specific mouse mAb.
The antibody was prepared as described in
Experimental procedures Purified EF-P
pro-tein was subjected to isoelectric focusing
run in one dimension followed by SDS
elec-trophoresis in the second dimension using
conditions described previously [23] (B)
Im-munoblots of polyribosomes treated with
anti-(EF-P) mAb The optical density profiles
of fractions collected after sucrose-density
gradient centrifugation of polyribosomes are
shown The position of the 70S, 50S and
30S particles is indicated by arrows An
immunoblot of EF-P, associated with
differ-ent fractions of the ribosome, is shown
below the ribosome profile (C) Binding of
EF-P to different polyribosome fractions.
The density of the immunoblots was
deter-mined, as was the area of each peak The
ratio of the density, which is related to
the amount of EF-P bound, to the area of
the peaks, which is related to the total
amount of the ribosome population in that
region of the polyribosome profile, is
plotted as a function of the percentage of
EF-P protein bound to each fraction.
Table 1 EF-P modification MS analysis of tryptic fragments of EF-P protein was carried out as described in Experimental procedures Table 1 provides information regarding the quality of the search match, including the acquired MS ⁄ MS scan numbers, final candidate search cross-correlation (X-corr) score, the normalized delta cross-correlation score (indicating the difference between the top-ranked and second best match), the preliminary database (SIMS) search score, the matched protein identity, the matched peptide sequence with putative modi-fication sites indicated with an @ symbol adjacent right hand to the target residue, and the predicted modimodi-fication mass The average modifi-cation mass, and SD, is also shown Modifimodifi-cation mass, 143.77 ± 0.15.
Scan
X-corr
SIMS score Protein
Sequence (@=modification)
Modification mass
Trang 4sulfate (DMS), in the presence of borohydride and
aniline, to score for A, C and G modifications in the
presence or absence of EF-P, and probed the 16S and
23S rRNA These results guided our further
chemi-cal probing with kethoxal (Ke),
1-cyclohexyl-3-(2-mor-pholinoethyl)carbodi-imidemetho-p-toluenesulfonate
(CMCT) and diethyl pyrocarbonate (DEP)
Recogni-tion sites were identified by primer extension analysis
with reverse transcriptase and several synthetic
deoxy-oligonucleotide primers The position of the modified
bases was detected by stops or pauses in the progress
of reverse transcription of the modified rRNA
tem-plate [20,21,24] Artifact bands, presumably arising
from nicks in the template rRNA or from strong
sec-ondary structure features, were distinguished from
sites of chemical attack by their occurrence in
tran-scripts using unmodified control rRNA, which had
otherwise been subjected to identical treatment (data
not shown)
Footprinting experiments were performed with the
intact 70S ribosomes in the presence or absence
of EF-P protein using probes complementary to
16S rRNA As shown in Fig 2, EF-P markedly
pro-tects G527, U534 and G537 against CMCT treatment
of 70S ribosomes Also, the reactivity of U531 is
enhanced by this treatment CMCT reacts slowly with
G residues [21]; however, DMS protection of this
region confirmed these results (data not shown) This region occurs near the G526 streptomycin binding site adjacent to the A-site of the 30S subunit, close to the S12 and mRNA binding sites [25]
Footprinting experiments were also performed on the 23S rRNA modified in the presence or absence of the ribosome-bound EF-P protein As shown in Fig 3, EF-P protects A2564, G2524, C2507, G2505, G2502
Table 2 Stoichiometric binding of EF-P to 70S ribosomes and to
30S and 50S subunits EF-P protein was labeled as described in
Experimental procedures Approximately 240 pmol labeled EF-P
were added to 140 pmol 70S ribosomes and incubated for 10 min
at 37 C prior to loading the proteinÆribosome complex on the
gradi-ents The samples were centrifuged in a 0–40% sucrose gradient
in 10 m M MgCl2, 10 m M Tris, HCl, pH 7.4 and 30 m M NH4Cl for
21 h at 4 C Samples were collected and the radioactivity and
A260values were determined.
a [ 14 C] EF-P bound ⁄ 70S, 50S or 30S.
Fig 2 Primer extension analysis of CMCT-modified 16S rRNA in
the presence (+) or absence (-) of EF-P protein was conducted as
described in Experimental procedures The positions of chemical
attack were determined using reverse transcriptase and a
22-nucle-otide cDNA 5¢-AGATGCAGTTCCCAGGTTG-3¢ primer
complemen-tary to bases flanking the G526 streptomycin binding site A, T, G,
C are dideoxy sequencing lanes Experiments were performed in
triplicate Ke and DMS treatment of the ribosomes verified the
location of the protected bases (data not shown) The data indicate
that EF-P protects the 16S rRNA near the A-site (see text).
Trang 5and G2494 in the 23S rRNA against DMS treatment
of the ribosome Significantly, the reactivity of A2572,
G2570, G2550 and C2498 is enhanced by the
interac-tion of EF-P with 70S ribosomes It is clear from the
data that EF-P markedly protects domain V of the
50S subunit, which is associated with the
peptidyl-transferase functions of the ribosome
Most important to the function of the EF-P protein
is the fact that most of the molecule’s interactions
occur at the aminoacyl-tRNAs (A-site) Indeed, one
set of bases protected by interactions of EF-P with the
70S ribosome occurs near the A-loop of the
peptidyl-transferase In addition, the protected bases, A2564 and G2524, map adjacent to the A-site on the 50S sub-unit near the ribosomal 530 loop that is involved in decoding by EF-TuÆGTPÆaminoacyl-tRNA complex, the T-site [26,27] One of the weakly protected bases, G537, also maps to the T-site of the 30S subunit It is possible that the T-site represents an alternate weak binding site for EF-P (Fig 5)
To further verify whether EF-P affects the ribosomal A-site, the fMet–tRNA was bound under conditions known to bind aminoacyl-tRNAs to either the ribo-somal A-site or the P-site The reaction was conducted
at 30C in the presence of GTP under conditions that foster spontaneous translocation [28] As shown in Fig 4A,C,D, GTP markedly stimulates the EF-P-dependent synthesis of fMet–puromycin when the fMet–tRNA is bound to the A-site This effect is greatly diminished when fMet–tRNA is bound under conditions that bind it to the presumed P-site (Fig 4B) The oxazolidinone III antibiotic, which impedes binding of fMet–tRNA to the P-site and inhibits translocation [29], interferes with the action of EF-P by preventing the fMet–tRNA substrate from re-binding to the P-site [29] Oxazolidinone III has little effect when fMet–tRNA is pre-bound to the P-site (Fig 4B), probably because fMet–tRNA out-competes oxazolidinone III on the P-site To learn whether fMet–tRNA binding is affected by the action of GTP, fMet–tRNA was bound in the presence of GTP and EF-P for 20 min at 30C followed by incubation with puromycin As shown in Fig 4C, the reaction proceeds quantitatively The controls show that the reaction decreases by 70% when fMet–tRNA is bound to the ribosome in the absence of GTP and EF-P, and these reagents are added during the second reaction with puromycin (Fig 4D) Thus, EF-P may interact with the A-site of the ribosome and may be required to accommodate fMet–tRNA in the P-site of the 70S ribosome
A computer-simulated structure of the 70S ribosome
of E coli was used to fit the protected bases of the EF-P protein relative to the A-, P-, and E-sites occu-pied by their respective tRNAs as well as the mRNA-binding sites (Fig 5) As shown in Fig 5, there appear
to be two possible binding sites for the protein One
of these occupies the 50S and 30S A-site, whereas the second, weaker binding site, appears to occur on the adjacent T-site of the 70S ribosome
Discussion
Biochemical studies have not revealed any effect of EF-P on the initiation reaction Thus, EF-P is not
Fig 3 Primer extension analysis of DMS-modified 23S rRNA in
the presence (+) or absence (–) of EF-P protein The positions of
chemical attack were determined using reverse transcriptase and a
22-nucleotide cDNA primer 5¢-TCTCCAGCGCCACGGCAGATAGG
GACC-3¢, complementary to domain V of the 23S rRNA as
described in Experimental procedures [20,21,24] A, T, G, C are
di-deoxy sequencing lanes Experiments were performed in triplicate.
The data indicate that EF-P protects domain V on the 23S rRNA,
which is close to the A-site of the peptidyltransferase center (see
text).
Trang 6required for the binding of fMet–tRNA in the presence
or absence of the initiation factors, IF-1, IF-2 or IF-3
[1,7,12] However, EF-P stimulates the initial rate of
translation programmed by poly(rU) when synthesis is
initiated by N-acetyl-Phe-tRNA [12] in the absence of
initiation factors Under these conditions, EF-P does
not stimulate the synthesis of poly(Phe) from
Phe-tRNA dependent on EF-Tu and EF-G [12] Thus, the
EF-P protein may not be required for initiation on
30S subunits or for elongation of protein chains EF-P
is required for synthesis of several fMet-inititated
di-peptides [8,12] Therefore, EF-P may function in the
formation of the initial peptides in the protein
sequence Consistent with this idea is the fact that
EF-P is found bound to native polyribosomes as well
as to 70S, 50S and 30S particles (Fig 1B,C) The
EF-P protein occurs in 0.1 copies per ribosome and
may dissociate from ribosomes, perhaps after synthesis
of the first peptide bond [30]
Here, we present the results of chemical protection footprinting analysis that provide clues concerning the nature of the in vitro associations between rRNA molecules and the EF-P protein The entire length of the exposed 16S and 23S rRNA molecules on the 70S ribosome was probed with DMS, Ke, DEP and CMCT in the presence or absence of EF-P A selected number of bases is protected from chemical probes
by the complex In several regions of the rRNA mole-cules, the complex enhanced the reactivity of specific bases, which is interpreted as being due to protein-dependent conformational changes in the rRNA [20] The evidence presented here indicates that the elon-gation factor, EF-P, binds predominantly to the A-site
of 70S ribosomes A possible second weak binding site
Fig 4 Effect of binding fMet–tRNA to the A- or to the P-site on the activity of EF-P f[ 35 S]Met–tRNA (36 pmol) was incubated with 70S ribosomes (20 pmol) for 20 min at
30 C as described previously [22,28] using 4.8 m M (A) or 7.2 m M Mg(Ac2) (B) for bind-ing, presumably to either the A- or the P-site
of the ribosome, respectively EF-P (20 pmol) and puromycin (1 l M ) were added and the reaction was continued at 30 C for
5 min in the presence or absence of 0.1 m M GTP and, where indicated, 50 l M oxazolidi-none III (oxo) (C) EF-P and GTP were added during formation of the fMet–tRNAÆribo-some complex (first reaction) and the reac-tion was carried out for 20 min at 30 C, prior to the addition of puromycin (second reaction) (D) The initiation complex was formed without GTP or EF-P for 20 min at
30 C, and GTP and EF-P were added with puromycin and were incubated for 5 min
at 30 C.
Trang 7for the EF-P protein occurs proximal to the A-site on
the ribosomal T-site that is the recognition center for
the EF-TuÆGTPÆaminoacyl-tRNA on the 30S and
50S subunit The footprints on the 16S rRNA reside
close to the mRNA binding site on the neck of the
30S subunit, adjacent to the G526 streptomycin and
the ribosomal protein S12 binding sites (Figs 2 and 5)
Interestingly, the structural paralog of EF-P, eIF5-A,
which shares strong sequence homology with
domains I and II of EF-P, binds to certain mRNAs in
a hypusine-dependent manner [31] Our MS analysis of
EF-P indicates that Lys34 of the protein is modified
by spermidine, which is a precursor of hypusine in the
eIF-5A protein [32] The model in Fig 5, in which the
orientation of the spermidine residue of EF-P is facing
the mRNA-binding pocket of the 30S subunit is
con-sistent with this result
The EF-P footprints extend into the 50S subunit
and reside close to the L7⁄ L12 stalk The 50S T-site
harbors the ribosomal L12 protein (GAR), which
occurs in an open state when the A-site is empty or
filled with tRNA [33], but closes upon contact with the
EF-Tu ternary complex [33] It is possible that EF-P
binds to the T-site in order to maintain a closed A-site
This may insure that the fMet–tRNA is accommo-dated on the P-site of the 70S ribosome prior to the proof-reading that occurs with the EF-Tu ternary com-plex and the ribosome
The EF-P protein binds stoichiometrically to the ribosome and stimulates accommodation of fMet– tRNA, presumably to the 70S P-site Thus, it is likely that the protein binds to more than one site on the ribosome depending on the course of synthesis Most bases protected upon binding of EF-P to the 70S ribo-some appear to reside on the A-site of the 30S and 50S subunits (Figs 3 and 5)
It is noteworthy that EF-P protects bases that occur at the A-site of the 50S subunit on 70S ribo-somes (Figs 3 and 5) This is consistent with the
EF-P protein being a competitive inhibitor of puromycin [34], an antibiotic known to act at the A-site of the ribosome [35] Reconstitution experiments, with core particles of the 50S subunit lacking specific 50S pro-teins, revealed that L16 is essential for the action of the EF-P protein [36] The crystal structure of the ribosome, with bound aminoacyl-tRNA, indicates that L16 occurs at the A-site of the 50S subunit [37] Furthermore, the L16 protein binds aminoacyl-tRNA [37] (and unpublished observations) The tRNA-like shape of the EF-P protein is in keeping with its interactions near the A-site of the 70S ribosome and its subunits The binding of EF-P at the A-site may have important consequences for proper decoding of the mRNA transcript For example, by binding to the A-site, EF-P may prevent the incorrect position-ing of the fMet-RNA into the A-site of the 50S sub-unit
The binding site of EF-P to domain V is of special importance because EF-P is predicted to enhance elon-gation by influencing in some manner the peptidyl-transferase center [12] Several bases indicated in the protection analysis (Fig 3) demonstrate that EF-P interacts close to the A-site of domain V of the 23S rRNA The X-ray diffraction patterns of the 70S ribosomes of Thermus thermophilus, at 5.5 A˚ reso-lution, reveal that the binding site of the three tRNAs
is adjacent to the binding site of the elongation factors [37] Thus, the incoming aminoacyl-tRNAÆGTPÆEF-Tu complex must be adjusted to the A-site in a position where a peptide bond can be formed By being at the A-site, EF-P may prevent the spurious entrance of aminoacyl-tRNAs, or of deacyl-tRNAs, which do not occur in the ternary complex with EF-Tu, from prema-turely entering the A-site prior to the proofreading steps which precede accommodation of the incoming aminoacyl-tRNA into the peptidyltransferase active site
Fig 5 Computer-simulated 3D structure of the 70S ribosome of
E coli showing the approximate location of the EF-P-binding site on
the 70S ribosome Bases protected against chemical modification
by interactions with EF-P are shown in red; bases enhanced by the
interactions with EF-P are shown in yellow The positions of the
exit (E) and peptidyltRNA (P) site on the 70S ribosome are shown.
EF-P (light green) clearly binds near the A-site of the 30S and 50S
subunits, as well as to the T-site of the ribosome The probable
spermidine-binding site (on Lys34 of EF-P protein) is shown in
orange and points towards the 30S subunit (see text) The
coordi-nates for the E coli ribosome were from Schuwirth et al [43] The
protections observed were also found to neighbor the A-site of the
70S ribosomes of T thermophilus determined by X-ray diffraction
at the 5.5 A ˚ resolution [37].
Trang 8We suggest that EF-P interacts with the A-site of
the peptide-bond-forming center of the large ribosomal
subunit and with the decoding center of the small
sub-unit Thus, the binding site may span both subunits
The oligonucleotide domain of the EF-P protein,
which harbors spermidine, may bind near the
mRNA-binding site (Fig 5), while the hydrophobic residues
around the central loop of the molecule might be
adja-cent to the peptidyltransferase adja-center Most important
to the function of the EF-P protein is the fact that the
interactions of the molecule are in close proximity to
the peptidyltransferase center of domain V near the
positions of the A-site 3¢-CCA-termini of the bound
tRNAs (Figs 3 and 5)
The footprints of the EF-P protein on the 16S
rRNA are adjacent to the G526 streptomycin-binding
site [22,35] and the ribosomal protein S12 (Figs 2 and
5) Streptomycin is a potent inhibitor of
EF-P-medi-ated synthesis of peptide bonds [12] Streptomycin
inhibits translation by increasing the error rate of
syn-thesis and interfering with the proofreading
mecha-nisms of the ribosome [25,35]
When a cognate tRNA binds to the A-site, the
30S subunit undergoes a conformational change from
an open to a closed form [25] Some mutations that
affect accuracy either prevent or induce this
conforma-tional transition [25] Streptomycin, for example,
stabi-lizes the closed form and induces errors in translation
By contrast, certain mutations in S12 to streptomycin
resistance or dependence destabilize the closed form
[25]
Thus, by binding near the A-site, EF-P may prevent
the aminoacyl-tRNA in the
EF-TuÆGTPÆaminoacyl-tRNA complex from prematurely entering the A-site
prior to the proofreading functions carried out by the
ribosome [25–27] This interaction would also help
poise the fMet–tRNA on the P-site of the 50S subunit
and may also result in increased accuracy of
amino-acyl-tRNA selection Hydrolysis of GTP results in
ejection of EF-TuÆGDP from the ribosome and
accom-modation of the aminoacyl-tRNA into the A-site
[26,27] A-site-bound aminoacyl-tRNA is predicted to
displace the EF-P protein from the ribosome Thus,
one EF-P molecule would be used for each successfully
initiated round of translation
The interactions of EF-P with the ribosome are
likely to result in rearrangements of the ribosomal
subunits that are essential for the transition between
the initiation and elongation stages of protein
synthe-sis The conservation of this protein throughout
spe-cies and its obligatory requirement during synthesis
befit the essential nature of these functions in
transla-tion
Experimental procedures
Materials The materials used were as described in Xu et al [5] and [29]
RNA modification and primer extension analysis The 70S ribosomes (8 pmol) were modified using DMS, DEP, Ke or CMCT in the presence or absence of 20 pmol EF-P protein Adenines and cytosines were determined as described previously [20,21] The N-7 position of guanine was detected with DMS using sodium borohydride and ani-line to induce strand scission [21] The DNA sequence was performed by a modification of the double-strand dideoxy chain termination method of Sanger et al with modified T7 DNA polymerase and [35S]dATP[aS] [20,21,24]
Effect of EF-P binding on the chemical protection footprints of 70S ribosomes
To locate the bases in 16S and 23S RNA on 70S ribosomes that are in contact with EF-P, EF-P was first bound stoi-chiometrically to the ribosome Each of the synthetic oligo-nucleotide primers was annealed to the rRNA in the complex and was extended with reverse transcriptase in the presence of [35S]dATP[aS] and the other three (unlabeled) deoxynucleotide triphosphates The labeled DNA tran-scripts were resolved on a DNA sequencing gel along with four dideoxy sequencing lanes on the same gel to help iden-tify the modified bases The entire length of the 16S or 23S rRNA on the 70S ribosomes was probed with DMS in the presence or in the absence of the EF-P protein Primers complementary to sequences at 200-bp intervals were then annealed to the ribosome [20] Based on the results with DMS, several different primers were utilized to further study the specificity of the regions protected against chemi-cal attack by Ke, CMCT and DEP Primers to the 30S sub-unit spanned the streptomycin binding sites at G526 and at A915 [25] Two additional primers were used to probe the decoding region of the 16S rRNA Two primers were used
to probe domain II of 23S rRNA; three primers were used
to probe domains IV and V and one primer was used to probe domain VI of the 23S rRNA molecule
Preparation of 70S ribosomes, 30S and 50S subunits
Ribosomes and subunits were isolated from E coli
MRE-600 mid-log cells at 4C as described previously [22] Briefly, cells (100 g) were broken by grinding with 100 g alumina (Alcoa Inc., Pittsburgh, PA, USA) and suspended
in buffer A (10 mm Tris⁄ HCl, pH 7.4, 30 mm NH4Cl,
1 mm dithiothreitol, 6 mm Mg(Ac)2), DNase (RNase-free;
Trang 91.0 lgÆmL)1) was added and the mixture incubated for
5 min at 4C Unbroken cells and debris were removed by
two successive centrifugations for 20 min at 30 000 g The
resulting supernatant was centrifuged at 78 000 g for 18 h
and the ribosomal pellets were suspended in buffer A
con-taining 0.5 m NH4Cl and the crude ribosomal suspension
was gently stirred for 2 h The crude ribosomal suspension
was layered on sucrose density gradients (0–40% sucrose in
buffer A) and centrifuged at 78 000 g for 18 h Fractions
containing the 70S ribosomes were combined and were
cen-trifuged for 24 h at 60 000 g The ribosomal pellets were
suspended in 2–3 mL buffer A containing 0.5 m NH4Cl, the
suspension was stirred for 2 h at 4C and was subjected to
a second 0–40% sucrose density gradient centrifugation
step as above at 64 000 g Absorbance at 260 nm was used
to identify the ribosomal fractions and the 70S peak was
pooled and centrifuged at 37 000 g for 18 h Ribosomes
were collected and suspended in buffer A at a concentration
of 20 mgÆmL)1and stored at)80 C
Isolation of EF-P
The EF-P protein was assayed based on its ability to
stimu-late ribosomes to synthesize fMet–puromycin in the
pres-ence of 1.0 lm puromycin and the abspres-ence of organic
solvents [1] using f[35S]Met–tRNA bound to ribosomes as
described previously [22,38]
The EF-P protein was purified as described previously
[12] Briefly, ribosomes were isolated as described above
and washed once with 0.5 m NH4Cl in buffer A containing
6 mm Mg(Ac)2 The ribosomal wash, containing the EF-P
protein, was sequentially purified first through a column of
QAE–Sepharose, followed by hydroxylapatite, Sephacryl
S-300 and Mono-Q columns using FLPC Fractions were
assayed after each step to locate the protein The purified
protein was concentrated and was stored at)80 C Protein
concentration was determined using the method described
by Bradford [39]
Preparation of mouse mAbs to EF-P
mAbs to the purified EF-P protein were raised using nude
mice This study received ethical approval by the University
of Toronto Animal Care Committee, which is in full
com-pliance with the Guidelines of the Canadian Council on
Animal Care and the Regulations of the Animals for
Research Act Aliquots of the EF-P (1 mgÆmL)1) in
buf-fer A were injected into the mice intravenously The
immu-nization schedule was 5 lg of the protein on day 1, 10 lg
on day 8, 20 lg on day 12 and 25 lg on day 16 After a
two-week rest, mice received a final 25 lg injection of the
EF-P protein Three days after the final injection, blood
was obtained through the orbital vein Three days after
this, the cells were harvested and fused with HAT-sensitive
mouse myeloma cells The samples were tested first for
anti-body production using dot blots Positive clones were ana-lyzed on western blots
Radioactive chemical labeling of the EF-P protein The EF-P protein (700 pmol) was dissolved at 1C in 0.1 m Na borate buffer (pH 9.0), 300 mm KC1 and 5 mm b-mercaptoethanol The [14C]formaldehyde (50 mCiÆmm)1) was added in the above buffer to the EF-P protein, mixed for 2 min prior to the addition of 8 lg Na borohydride to stop the reaction The labeled EF-P was dialyzed against
10 mm HEPES buffer, pH 7.4, 10 mm b-mercaptoethanol
to eliminate the unreacted [14C] formaldehyde
Tandem MS Proteomic analysis of the samples was performed by microcapillary electrospray LC-MS⁄ MS Briefly, an aliquot
of the EF-P protein was suspended in 100 mm
NH4HCO3⁄ 1 mm CaCl2 buffer, pH 8.5 and digested by trypsin overnight at 37C using immobilized trypsin Porous beads (PerSeptive Biosystems, Framingham, MA, USA) The digested peptides were then fractionated on a 7.5 cm (100 lm ID) reverse-phase C18 capillary column attached inline to a Thermo Finnigan LCQ-Deca quadrupole ion trap mass spectrometer The entire digested sample was loaded as described previously [40] and the peptides eluted
by ramping a linear gradient from 2 to 60% solvent B over
90 min Solvent A consisted of 5% acetonitrile, 0.5% acetic acid and 0.02% heptofluorbutyric acid and solvent B con-sisted of 80 : 20 acetonitrile⁄ water (v ⁄ v) containing 0.5% acetic acid and 0.02% heptofluorbutyric acid The flow rate
at the tip of the needle was set to 300 nLÆmin)1by program-ming the HPLC pump and use of a split line The mass spectrometer cycled through four scans as the gradient pro-gressed The first event was a full mass scan followed by three tandem mass scans of the successive three most intense precursor ions Precursor ions (400–2000 m⁄ z) were sub-jected to data-dependent, collision-induced dissociation with dynamic exclusion enabled The spectra were searched against UniProt protein sequences downloaded from the European Bioinformatics Institute using the sequest com-puter algorithm [41] Precursor mass tolerance was set to
3 Da (with daughter mass ion tolerance set to the default of 0), enabling fully tryptic enzyme status, with single site missed cleavages tolerated The statistical probability of each primary match was assessed using the statquest algo-rithm [42], with candidate peptides filtered using a strict 95% confidence cut-off to minimize false positives
Acknowledgments
We thank NSERC of Canada for financial support
We thank Jennifer Yang and Ivona Kozieradsky for
Trang 10excellent technical help during the initial stages of this
work We are most grateful to Drs A J Becker and
K Nierhaus for perceptive discussion of this study
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