From the Department of Biochemistry and Molecular
Biology, Louisiana State University Health Sciences Center, Shreveport,
Louisiana 71130-3932 and the ¶ Department of Biochemistry, School
of Medicine, Case Western Reserve University,
Cleveland, Ohio 44106-4935
Received for publication, July 18, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Eukaryotic translation initiation factor 4G-1
(eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA
helicase eIF4A, one in the central domain and one in the COOH-terminal
domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did
not interfere with eIF4A binding to the central site. Interestingly, at
the same concentration of free eIF4A, more eIF4A was bound to an eIF4G
fragment containing both eIF4A sites than the sum of binding to
fragments containing the single sites, indicating cooperative binding.
Binding of eIF4A to an immobilized fragment of eIF4G containing the
COOH-terminal site was competed by a soluble eIF4G fragment containing
the central site, indicating that a single eIF4A molecule cannot bind
simultaneously to both sites. The association rate constant,
dissociation rate constant, and dissociation equilibrium constant for
each site were determined by surface plasmon resonance and found to be,
respectively, 1.2 × 105
M The initiation of translation of most eukaryotic mRNAs
involves the sequential recruitment of Met-tRNAi, mRNA, and the 60 S ribosomal subunit to the 40 S ribosomal subunit, catalyzed by
the various groups of initiation factors (1). One of the most highly
regulated steps is the recruitment of mRNA, which requires
recognition of the 5'-terminal m7GTP-containing cap and
3'-terminal poly(A) tract, unwinding of 5'-terminal secondary
structure, and binding to the 43 S initiation complex. These steps are
mediated by members of the
eIF41 group of initiation
factors (eIF4A, eIF4B, eIF4E, and eIF4G) as well as poly(A)-binding protein.
eIF4A is the prototypical member of the DEXD/H-box protein
family of nucleic acid helicases (2, 3). It functions as an
ATP-dependent, bi-directional RNA helicase and
RNA-dependent ATPase (4-7). The There are at least two genes for eIF4G in humans (12-14), wheat germ
(15), and yeast (16). In mammals, these are termed eIF4G-1 and
eIF4G-2.2 eIF4G serves to
colocalize initiation factors involved in mRNA recruitment to the
43 S initiation complex. It directly binds RNA (17-19),
poly(A)-binding protein (13), eIF4E (20, 21), the 40 S-binding protein
complex eIF3 (20), eIF4A (20, 22), and the eIF4E kinase Mnk1 (23, 24).
eIF4G in complex with these factors has the effect of bringing together
the 3'- and 5'-termini of the mRNA (via poly(A)-binding protein and
eIF4E), facilitates the binding of 40 S ribosomal subunit to mRNA
(via eIF3), and promotes unwinding of 5'-untranslated region secondary structure (via eIF4A).
Initial studies indicated that the NH2-terminal one-third
of mammalian eIF4G binds poly(A)-binding protein (13) and eIF4E (20,
21), the central one-third binds eIF3 (20), and the COOH-terminal
one-third binds eIF4A (20) and Mnk1 (23, 24). These portions of eIF4G
correspond with cleavage products of picornaviral proteases
(cpN (aa 1-634), cpC3 (aa 635-1041), and
cpC2 (aa 1042-1560), respectively; Ref. 20), suggesting
that they represent individual structural and functional domains of the
eIF4G molecule (see Fig. 1). The central domain has high amino acid
sequence homology to all eIF4G proteins, whereas the COOH-terminal
domain is poorly conserved (25). Additional studies revealed a second
eIF4A-binding site in the central domain (22). The central domain of
eIF4G is sufficient to catalyze cap-independent binding of ribosomes to
RNA (17) and cap-independent but 5'-end-dependent
translation in vitro (26) and in vivo (27). The
function of the COOH-terminal domain is less clear. Variant forms of
eIF4G containing both of the intact eIF4A-binding sites are more active
in formation of the 48 S initiation complex and stimulation of
cap-dependent translation than forms containing only one
site (28). This has led to the proposal that interaction of eIF4A with
the central domain is necessary for translation, whereas interaction of
eIF4A with the COOH-terminal domain plays a modulatory role (28).
A complex of initiation factors termed eIF4F can be isolated by
treatment of the 100,000 × g ribosomal pellet with 0.5 M KCl (29, 30). The eIF4F complex from mammalian cells
contains eIF4A, eIF4E, and eIF4G (29, 30), but the complex from wheat germ (31, 32), yeast (33, 34), and Drosophila (35) contains only eIF4E and eIF4G. Even in mammalian eIF4F preparations, the amount
of eIF4A varies, and passage of eIF4F over a phosphocellulose column
completely removes eIF4A (11, 36). The stoichiometries of the mammalian
eIF4F subunits have not been reported. The discovery of a second
eIF4A-binding site in eIF4G (22) raises the question of whether two
eIF4A molecules can bind simultaneously or whether a single eIF4A binds
to both sites, as suggested recently (28). Similarly, the affinities of
eIF4A to the two sites in mammalian eIF4G have not been reported. Since
binding of eIF4A to eIF4G serves not only to localize eIF4A in the
vicinity of the ribosome and mRNA but also enhances its intrinsic
helicase activity, we undertook an analysis of the two eIF4A-binding
sites in human eIF4G.
Materials--
m7GTP-Sepharose 4B, heparin-Sepharose
CL-6B, and a Mono Q HR 5/5 column were obtained from Amersham Pharmacia
Biotech. Econo-Pac® 10 DG disposable chromatography columns and the
Bio-Rad protein assay kit were obtained from Bio-Rad.
S-protein-agarose, S-protein-bacterial alkaline phosphatase conjugate,
and the plasmid pET-32a(+) were obtained from Novagen (Madison, WI).
Nickel nitrilotriacetic acid-agarose was obtained from Qiagen
(Chatsworth, CA). Bovine serum albumin was purchased from Pierce.
Isopropyl- Amino Acid and Nucleotide Nomenclature--
Several cDNAs
for eIF4G have been described that differ in the predicted length of
the NH2-terminal polypeptide sequences (12, 13, 37). The aa
and nt numbers used in the current work correspond to the larger form
of eIF4G that begins with MNTPSQ (13). In this numbering system, the
primary entero- and rhinoviral 2A protease cleavage site (38) is
located between Arg-641 and Gly-642 (nt 1823-1828) (see Fig. 1).
Fragments of eIF4G expressed from recombinant plasmids are named using
inclusive aa numbers. Thus, S-eIF4G(877-1078) would contain the eIF4G
sequence from aa 877 to 1078 fused at the NH2 terminus to
thioredoxin, a His6 sequence, and the S-peptide of RNase A
(which increases the molecular mass by ~20 kDa) (see Fig. 1). The
plasmid expressing this protein would be called pTS4G(877-1078).
Construction of Plasmids--
pTS4G(613-1560) was constructed
by inserting a restriction fragment from SmaI (nt 1749 in
eIF4G cDNA) to XhoI (polylinker) derived from pSK-HFCl
(39) between the EcoRV and XhoI sites of
pET-32a(+) and then deleting the sequence from the BglII
(polylinker) to BamHI (nt 1938 in eIF4G cDNA) sites.
pTS4G(613-1078) was constructed by deleting a fragment between
SpeI (nt 3334 in eIF4G cDNA) and SpeI
(polylinker) sites from plasmid pSK( Purification of Recombinant Proteins--
The eIF4G fragments
S-eIF4G(613-1560), S-eIF4G(613-1078), S-eIF4G(877-1078),
S-eIF4G(975-1078), S-eIF4G(1078-1560), and S-eIF4G(1317-1560) were
expressed in Escherichia coli strain BL21(DE3)pLysS
(Novagen) and purified by nickel nitrilotriacetic acid-agarose
chromatography. Cells were grown to A600 = 0.4 at 37 °C (typically 1-liter cultures except for S-eIF4G(613-1560)
and S-eIF4G(613-1078), for which 4-l cultures were grown), and then
expression was induced with isopropyl-
In the case of S-eIF4G(613-1078), it was necessary to perform an
additional purification step on heparin-Sepharose CL-6B. The eluate
from nickel nitrilotriacetic acid-agarose was diluted with an equal
volume of buffer B0 (20 mM Tris-HCl, 2 mM EDTA, 5 mM
eIF4G(642-1560) (used as a standard) and eIF4G(642-1078) were
produced by incubation of S-eIF4G(613-1560) and S-eIF4G(613-1078), respectively, with recombinant Coxsackievirus 2A protease at 50 µg/ml
for 1 h at 4 °C (41). In the case of eIF4G(642-1560), the
resultant COOH-terminal portion was further purified from the
S-peptide-tagged NH2-terminal portion by adsorption of the latter to S-protein-agarose.
Before performing binding experiments with S-protein-agarose, purified
eIF4G fragments and eIF4A were passed over desalting Econo-Pac® 10 DG
disposable chromatography columns to replace the buffer with buffer
C150 (20 mM HEPES-KOH, 150 mM KCl,
2 mM eIF3, eIF4A, and eIF4F Purification--
Purification and
14C-labeling of eIF4A by reductive methylation was
performed as described previously (42). The particular batch of
[14C]eIF4A used in these experiments was labeled to a
specific activity of 187 cpm/pmol. eIF3, eIF4A, and eIF4F were purified
from the ribosomal high salt wash of rabbit reticulocyte lysate by
m7GTP-Sepharose and Mono Q chromatography (43). The eIF4A
peak was rechromatographed on Mono Q with a shallower salt gradient. The eIF3 peak from the initial Mono Q chromatography was further purified by gel filtration on a SW300 column (Waters, Milford, MA) in
buffer C150 plus 5% (v/v) glycerol.
Protein Binding Assays on S-protein-agarose--
Binding of
S-eIF4G fragments with eIF3 and eIF4A was performed using
S-protein-agarose. After 40 min of preincubation of S-eIF4G fragments
with eIF3 or eIF4A on ice, proteins were mixed with at least a 10-fold
molar excess of S-protein-agarose and incubated for 2 h in buffer
C150 containing 1% milk proteins at 4 °C. The resin was
washed 4 times with 200-µl aliquots of buffer C150. Proteins were eluted in SDS-electrophoresis buffer and analyzed by
SDS-PAGE (44), with detection by Coomassie Blue staining, Western
blotting, or autoradiography.
Antibodies--
Mouse monoclonal anti-eIF4A antibodies were a
gift from Dr. Hans Trachsel, Bern, Switzerland. Rabbit anti-human eIF4G
peptide 9 (aa 809-822) antibodies were produced as described
previously (20).
Western Blotting--
For immunoblotting, proteins were
transferred after SDS-PAGE to an Immobilon-P membrane (Millipore,
Bedford, MA) using a Mini Trans-Blot cell (Bio-Rad). The membrane was
incubated with a 1:1000 dilution of the primary antibodies in 5% milk
proteins in buffer E (20 mM Tris-HCl, 150 mM
NaCl, and 0.05% Tween 20, pH 7.5) for 1 h at room temperature,
washed three times for 10 min with buffer E, and incubated with
secondary anti-mouse or anti-rabbit antibodies conjugated with alkaline
phosphatase (Vector Laboratories, Inc., Burlingame, CA) at a dilution
of 1:2000 in 5% milk proteins in buffer E for 1 h at room
temperature. Blots were developed with the nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl-phosphate color development substrate
(Promega, Madison, WI).
Quantitation of Binding Data--
Quantitation of eIF4G
fragments and eIF4A separated by SDS-PAGE was performed using a
ScanMaker III laser densitometer (Microtek) and ImageQuant software,
Version 3.3 (Molecular Dynamics). Experimental data were compared with
standard curves consisting of purified S-eIF4G fragments and eIF4A for
which the concentration has been determined with the Bio-Rad protein
assay kit. Curve fitting was performed using SigmaPlot software,
Version 4.01 (SPSS, Inc.). In cases of eIF4A binding with
S-eIF4G(613-1078) or S-eIF4G(1078-1560), the data were fit with an
equation describing the Langmuir isotherm,
An equation that takes into account two dissimilar binding sites (45)
was used in titrations involving S-eIF4G(613-1560),
SPR Analysis of S-eIF4G·eIF4A Interactions--
SPR was
carried out using a BIAcore 2000 instrument (Biacore, Inc., Piscataway,
NJ). eIF4A was immobilized on a research grade CM5 sensor chip using
the amino coupling kit supplied by the manufacturer in 10 mM sodium acetate, pH 4.5. The surface density of
immobilized eIF4A was 200-300 RU. One RU corresponds to an immobilized
protein density of 1 pg/mm2 (46). The portion of the sensor
chip in the first flow cell, used as a control, was subjected to
activation and blocking in the same way as the eIF4A-containing cells
but without added protein. The signals generated in the control flow
cell were subtracted from the experimental signals to correct for
refractive index changes and nonspecific binding.
All kinetic experiments were carried out in buffer D at 25 °C and a
flow rate of 50 µl/min for S-eIF4G(613-1078) and S-eIF4G(1078-1560) and 20 µl/min for S-eIF4G(975-1078). At least five different
concentrations of each S-eIF4G fragment were injected for each
experiment. The first injection contained buffer without the S-eIF4G
fragment. Between injections, the surface was regenerated with buffer F (20 mM HEPES-KOH, 500 mM KCl, 3 mM
EDTA, 0.1% Tween 20, and 2 mM
Kinetic and equilibrium constants were calculated using the
curve-fitting facility of the BiaEvaluation software, Version 3 (Biacore, Inc.). Binding data were globally fit to the 1:1 Langmuir binding model (A+B Definition of eIF4A-binding Sites on eIF4G--
Two eIF4A binding
domains have been described for mammalian eIF4G, one in the central
domain (22) and the other in the COOH-terminal domain (20) (Fig.
1). Evidence has recently been presented
that the central eIF4A-binding site is located between aa 672 and 970 (28). To study these two binding sites separately, we prepared various
recombinant portions of eIF4G fused to an NH2-terminal tag
containing the S-peptide of RNase A (Fig. 1). [14C]eIF4A
was incubated with each of these S-tagged eIF4G fragments, and the
resultant complex was captured on S-protein-agarose. Material bound to
the resin was eluted, subjected to SDS-PAGE, and analyzed for the eIF4G
fragment by Coomassie Blue staining (Fig.
2A) and for eIF4A by
autoradiography (Fig. 2B). S-eIF4G(613-1078) and S-eIF4G(1078-1560) were observed to bind eIF4A (lanes 1 and
2), but the other S-eIF4G fragments did not (lanes
3-5).
Since both eIF4A and eIF4G are capable of binding RNA, there was a
possibility that the observed binding was due to an RNA linker.
Therefore, the experiment was repeated after treatment of the proteins
with micrococcal nuclease to remove any E. coli RNA that may
have copurified with the recombinant proteins. The results were not
altered from those seen in Fig. 2 (data not shown), indicating that the
binding occurs in an RNA-independent manner.
eIF4A and eIF3 Do Not Compete for Binding to the Central Domain of
eIF4G--
The central domain binds eIF4A as well as eIF3 (20, 22). To
test whether this domain can bind eIF4A and eIF3 simultaneously, we
performed a competition experiment. Reaction mixtures contained S-eIF4G(613-1078), a 5-fold molar ratio of [14C]eIF4A to
S-eIF4G(613-1078), and a 2-, 5-, or 10-fold molar ratio of eIF3 to
S-eIF4G(613-1078) (Fig. 3, lanes
2, 3, and 4, respectively). After
incubation, the S-eIF4G(613-1078) and any bound proteins were captured
with S-protein-agarose. The results indicated that eIF3 was retained on
the S-protein-agarose (Fig. 3A, lanes 2-4). Furthermore, there was no decrease in eIF4A binding when increasing amounts of eIF3 were added, whether measured by Coomassie Blue staining
(Fig. 3A, lanes 2-4) or autoradiography (Fig.
3B, lanes 2-4). There was no nonspecific binding
of eIF4A or eIF3 to the resin in the absence of the eIF4G fragment
(Fig. 3, lane 8).
Another series of reaction mixtures contained S-eIF4G(613-1078), a
5-fold molar ratio of eIF3 to S-eIF4G(613-1078), and a 2-, 5-, or
10-fold molar ratio of [14C]eIF4A to S-eIF4G(613-1078).
Proteins were again captured with S-protein-agarose (Fig. 3,
lanes 5, 6, and 7, respectively). The results indicated that increasing amounts of eIF4A did not decrease eIF3 binding to S-eIF4G(613-1078) (Fig. 3A). The amount of
bound [14C]eIF4A did not increase as the concentration of
added [14C]eIF4A was increased because at all points, it
was in molar excess to S-eIF4G(613-1078). These data show that there
is no competition between eIF3 and eIF4A for binding to the
S-eIF4G(613-1078) and, therefore, that the central domain of eIF4G can
bind eIF4A and eIF3 simultaneously.
eIF4G(613-1078) and eIF4G(1078-1560) Each Bind eIF4A with a
Stoichiometry of 1:1 but with Different Affinities--
To estimate
the stoichiometry and equilibrium binding constants of eIF4A with
S-tagged eIF4G fragments, we carried out titration experiments using
S-protein-agarose. S-eIF4G(613-1078) was incubated at a concentration
of 0.3 µM with either no eIF4A or with a 0.2-5-fold molar ratio of eIF4A. After adsorption to S-protein-agarose, the material bound to the resin was eluted and analyzed by SDS-PAGE, Coomassie Blue staining, and autoradiography (Fig.
4A). One reaction mixture
containing eIF4A but no eIF4G fragment indicated that nonspecific
binding of eIF4A was negligible (lane 12). The binding data
were quantitated using standard curves, and a nonlinear least squares
fit of the experimental data was performed using Equation 1 (Fig. 4B).
The results indicated that the number of eIF4A-binding sites on
S-eIF4G(613-1078), n, was 1.1 and that the equilibrium dissociation constant, Kd, was 96 nM
(Fig. 4B). A linear transform of the data indicates the
presence of a single binding site (Fig. 4B,
inset).
A similar experiment was performed with S-eIF4G(1078-1560). The
S-eIF4G(1078-1560) was incubated at a concentration of 0.6 µM with a 0.5-10-fold molar ratio of eIF4A to
S-eIF4G(1078-1560) (Fig. 4C). The data were quantitated as
described above. The nonlinear least-squares fit yielded
n = 1.1 and Kd = 660 nM,
indicating that eIF4A binds S-eIF4G(1078-1560) with a stoichiometry of
1:1 (Fig. 4D). Scatchard analysis confirmed the existence of
a single binding site on S-eIF4G(1078-1560) (Fig. 4D,
inset). These and several other similar experiments
indicated that, within experimental error, the stoichiometry of eIF4A
binding to both the central and COOH-terminal sites was 1:1.
Stoichiometry of Binding of eIF4A with eIF4G(613-1560)--
The
forgoing result indicated that one molecule of eIF4A could bind to
either the central or COOH-terminal sites of eIF4G. The question then
arose as to whether binding to these sites is mutually exclusive. We
therefore titrated an eIF4G fragment containing both the central and
COOH-terminal sites [S-eIF4G(613-1560)] using the same methodology.
S-eIF4G(613-1560) was incubated at a concentration of 0.075 µM with eIF4A at a 1-16-fold molar ratio (Fig.
4E). Material bound to the resin was analyzed by
SDS-PAGE.
Unique values of n and Kd for each site
cannot be determined from empirical saturation data when there are two
dissimilar binding sites on the same molecule (45). We therefore
computed a theoretical curve from Equation 2 using the n and
Kd values for S-eIF4G(613-1078) (1.1 and 96 nM, respectively) and S-eIF4G(1078-1560) (1.1 and 660 nM, respectively) that were determined experimentally in
Figs. 4, B and D. The theoretical curve was similar to the experimental data, but several points fell above the
line (see below). In addition, the maximum eIF4A binding to S-eIF4G(613-1560) approached a stoichiometry of 2:1 (Fig.
4F). Binding of eIF4A to the S-eIF4G(543-1560) fragment was
also performed. In two separate experiments, the binding of eIF4A to
that fragment also approached a 2:1 stoichiometry (data not shown).
As expected, plotting the theoretical curve calculated from Equation 2
in the manner used to produce a linear transform of the data of Figs.
4, B and D, resulted in a curved line (Fig. 4F, inset). The fact that several of the points
fall above the theoretical curve means that the affinity of eIF4A for
an eIF4G fragment containing both sites is higher than predicted by
Equation. 2, which merely sums the individual contributions of the two
separate sites. This suggests that there is cooperativity between
sites, i.e. that the binding of eIF4A to one site increases
the affinity to the other. Further support for cooperative binding is
provided in the following experiment.
The Presence of Two eIF4A-binding Sites Enhances the Affinity of
eIF4A for eIF4G--
Cooperativity between sites would predict that an
eIF4G fragment containing both sites would bind more eIF4A than the sum of binding to fragments containing each separate site. To test this, we
individually incubated S-eIF4G(613-1078) (central site), S-eIF4G(1078-1560) (COOH-terminal site), and S-eIF4G(613-1560) (both
sites), each at 0.1 µM, with varying concentrations of
eIF4A. The eIF4G fragments and bound eIF4A were then captured with
S-protein-agarose and analyzed by Western blotting (Fig.
5). eIF4A incubated with a protein
consisting of all elements at the NH2 terminus of the S-eIF4G fusion proteins (thioredoxin, His6-tag, and S-tag)
but no eIF4G sequences was not retained on the resin (data not shown). At 0.39 µM eIF4A, the BR of eIF4A to S-eIF4G(1078-1560)
was only 0.02, whereas the BR to S-eIF4G(613-1078) was 0.65, which is
expected from the higher affinity of the latter. The predicted BR for
S-eIF4G(613-1560), if there is no cooperativity between sites, is the
sum of these, or 0.67. The observed BR, however, was 1.35, representing
a 2-fold enhancement. At 0.65 µM eIF4A, the cooperative
effect was not as high, which is to be expected since the sites were
more nearly saturated. The sum of the BR values for the individual
sites was 0.68, but the observed BR for S-eIF4G(613-1560) was 1.16, a
1.7-fold enhancement. Finally, at the highest eIF4A concentration, 1.3 µM, where both eIF4A-binding sites were nearly saturated,
the enhancement was only 1.2-fold. Thus, binding of eIF4A to an eIF4G fragment that contains both eIF4A-binding sites is more than additive. These observations support the idea of positive cooperativity between
the two sites.
Kinetic Measurements of eIF4A-eIF4G Interactions Using
SPR--
The assessment of binding properties by retention of
eIF4A·S-eIF4G complexes on S-protein-agarose represents a
nonequilibrium situation, because the agarose beads must be washed
several times with buffer not containing eIF4A to reduce nonspecific
binding. It is suitable for determination of stoichiometries because a theoretical maximal binding is approached with increasing eIF4A concentration. However, the technique produces a systematic
overestimate of Kd values (underestimate of binding
affinities) because the average eIF4A concentration during binding and
washing is less than the concentration during binding alone. We
therefore turned to SPR, a technique that is not subject to this
potential source of error, for the determination of binding constants.
eIF4A was immobilized on a sensor chip by the amino-coupling procedure,
and eIF4G fragments were passed over it. Binding of S-eIF4G(613-1078)
was measured over a range of concentrations from 3 to 100 nM (Fig. 6A).
Binding of S-eIF4G(1078-1560) was measured over 10 to 1000 nM (Fig. 6B). As a control, S-eIF4G(975-1078), which carries the same tags as two other eIF4G fragments but does not
contain an eIF4A-binding site (Fig. 2), was passed over the immobilized
eIF4A at concentrations ranging from 3 to 300 nM (Fig. 6C). The three sensograms confirmed that S-eIF4G(613-1078)
and S-eIF4G(1078-1560) each contain an eIF4A-binding site, whereas S-eIF4G(975-1078) does not. Thus, the NH2-terminal amino
acid sequence (thioredoxin, His6, and S-peptide) does not
interfere with the measurement.
The best fit to the experimental data for binding of S-eIF4G(613-1078)
and S-eIF4G(1078-1560) to eIF4A was provided with the simple 1:1
binding model (thin lines in Figs. 6, A and
B; see "Experimental Procedures"). The low values of the
average residuals (deviation of actual from theoretical) and
eIF4G(613-1078) and eIF4G(1078-1560) Compete for Binding to
eIF4A--
The presence of two eIF4A-binding sites on eIF4G evokes two
possible models regarding the domain or surface of eIF4A that binds
each site. In Model 1, suggested previously (28), a single eIF4A
molecule can bind simultaneously to both sites in eIF4G. This would
occur if two different surfaces of eIF4A were bound to the two sites.
This model is not contradicted by the finding of a 2:1 stoichiometry
with S-eIF4G(613-1560) (Fig. 4F), since at sufficiently
high eIF4A concentrations, a second eIF4A molecule could displace the
first, thus converting an eIF4A·eIF4G complex, with two contacts
between eIF4G and eIF4A, to a (eIF4A)2·eIF4G complex,
with a single contact between eIF4G and each of the eIF4A molecules. In
Model 2, the same surface of eIF4A binds to either site on eIF4G. In
this model, binding of eIF4A to one site on eIF4G prevents its
simultaneous binding to the other site. An alternative version of Model
2 that would predict the same results is that the two surfaces on eIF4A
overlap each other.
To distinguish between these two models, we performed a competition
experiment. One eIF4G fragment containing the COOH-terminal eIF4A-binding site and an S-tag, S-eIF4G(1078-1560), was incubated with [14C]eIF4A in the presence of increasing amounts of
an eIF4G fragment containing the central eIF4A-binding site but no
S-tag, eIF4G(642-1078) (the latter was produced by cleavage of
S-eIF4G(613-1078) with Coxsackievirus 2A protease. Then the S-tagged
eIF4G fragment was captured with S-protein-agarose (Fig.
7).
Two outcomes are predicted if Model 1 is correct. First, the untagged
eIF4G fragment would be retained on S-protein-agarose in a
"sandwich"
[S-protein-agarose·S-eIF4G(1078-1560)·eIF4A·eIF4G(642-1078)]. In Model 2, no untagged eIF4G fragment should be bound to the resin.
The second prediction is that addition of the untagged eIF4G fragment
should not decrease the amount of eIF4A bound to the S-protein-agarose
if Model 1 is correct. In fact, based on the observation of positive
cooperativity between the two eIF4A-binding sites (Figs. 4F
and 5), one would expect enhanced binding of eIF4A to the
S-protein-agarose. Model 2, on the other hand, predicts that the
untagged eIF4G fragment in solution would compete with the S-tagged
eIF4G fragment for binding with eIF4A, resulting in reduction of eIF4A
bound to the resin.
Reaction mixtures containing S-eIF4G(1078-1560), a 2-fold molar ratio
of [14C]eIF4A to S-eIF4G(1078-1560), and untagged
eIF4G(642-1078) in a 0-, 2-, 5-, and 10-fold molar ratio to
S-eIF4G(1078-1560) were incubated and subjected to S-protein-agarose
(Fig. 7, lanes 2, 3, 4, and 5, respectively). As shown in Fig. 7A, the
amount of S-eIF4G(1078-1560) retained on S-protein-agarose was
essentially constant, ranging from 2.9 to 3.1 pmol. The amount of
untagged eIF4G(642-1078) retained on the resin was much less, ranging
from 0.1 to 0.38 pmol (Fig. 7B, lanes 2-5).
Comparison of Fig. 7B, lane 3 (eIF4G fragment
treated with 2A protease) with Fig. 7B, lane 10 (the same molar amount as on lane 3, but the eIF4G fragment was not treated with Coxsackievirus 2A protease) shows that only 1.6%
of the input competitor (0.1 pmol versus 6.1 pmol) was
retained on the resin. Also, eIF4G(642-1078), a protein of about 50 kDa, was undetectable by Coomassie Blue staining (Fig. 7A,
lanes 2-5 and lanes 7-9). The eIF4A retained on
the resin was initially 0.98 pmol but decreased 6-fold to 0.17 pmol
with increasing competitor (Fig. 7C, lane
2-5).
As a control, one tube contained S-eIF4G(1078-1560),
[14C]eIF4A, untagged eIF4G(642-1078), and eIF3 (Fig. 7,
lane 6). If S-eIF4G(1078-1560) and eIF4G(642-1078) were to
make a sandwich via eIF4A, it would be possible to detect eIF3 bound to
the S-protein-agarose, since eIF3 binds to eIF4G(642-1078) (20).
However, there was no detectable eIF3 on the gel (Fig. 7A,
lane 6). Another set of controls contained untagged
eIF4G(642-1078) and [14C]eIF4A but no
S-eIF4G(1078-1560) (lanes 7-9). The amount of
eIF4G(642-1078) bound to the resin was low (0.03-0.46 pmol; Fig.
7B). Most significantly, there was no difference in the
amount of eIF4G(642-1078) bound to the resin in the presence or
absence of S-eIF4G(1078-1560) (Fig. 7B, lanes
3-5 versus 7-9), indicating that the
binding of eIF4G(642-1078) to the resin was nonspecific. All
these finding are consistent with Model 2 but not Model 1, indicating
that the two eIF4A-binding sites on eIF4G compete with each other for
binding to eIF4A and fail to form a sandwich with eIF4A.
In our earlier study on the domain structure of eIF4G, we
presented evidence that eIF3 was bound to the central domain, whereas eIF4A was bound to the COOH-terminal domain (22). A subsequent study
showed that eIF4A was also bound to the central domain (28). This
raised the question of the relationship between the eIF3- and
eIF4A-binding sites in the central domain. A previous study proposed
that these two sites overlapped (28). This suggestion was based on the
observation that mutations affecting eIF4A binding to the central
region of eIF4G also caused loss of eIF3 binding (22, 28). Here we show
that eIF3 and eIF4A do not compete for binding in the central domain,
which suggests that the eIF4A- and eIF3-binding sites are distinct.
The stoichiometries of mammalian eIF4F subunits have not been
previously determined. Furthermore, the relative amount of eIF4A in any
given preparation of rabbit eIF4F
varies.3 eIF4A is lost from
the mammalian eIF4F complex by phosphocellulose chromatography (36,
11), Mono Q chromatography (43), and ultracentrifugation of eIF4F on
sucrose gradients (20). For these reasons, merely measuring the
relative amount of eIF4F subunits in purified preparations would not be
expected to yield meaningful results. The approach we have taken here
is to measure the direct interaction of purified eIF4A and recombinant
eIF4G fragments. Titration of different S-tagged eIF4G fragments with
eIF4A demonstrated that eIF4G fragments containing each of the
individual eIF4A-binding sites bind eIF4A with a 1:1 stoichiometry, but
fragments containing both sites bind eIF4A with a 1:2 stoichiometry.
We showed that the affinities of the two eIF4A-binding sites are
different using two different methodologies, static binding on
S-protein-agarose and kinetic binding using SPR. Both methods indicated
that the central site has a higher affinity for eIF4A than the
COOH-terminal binding site. The two methods did not agree quantitatively, however, with apparent Kd values by
SPR being lower than those by S-protein-agarose. The SPR results are expected to be more accurate, since SPR measures binding and
dissociation instantaneously, whereas the S-protein-agarose method
measures only the protein bound after several washes to remove
nonspecifically bound protein. Given the propensity of eIF4F to lose
eIF4A during purification, it is not surprising that the apparent
affinities as determined by SPR are higher. The fact that the best fit
of the SPR data was provided by the 1:1 kinetic binding model provides further evidence for a 1:1 stoichiometry for each site.
The basis for the difference in affinities between the central and
COOH-terminal eIF4A-binding sites is revealed by the kinetic results.
Turnover of eIF4A occupancy of the central site is faster than that of
the COOH-terminal site. eIF4A associates with eIF4G(613-1078) about
24-fold faster than with eIF4G(1078-1560) (ka = 1.21 × 105 versus 5.1 × 103 M Our data suggest that the binding of two molecules of eIF4A to eIF4G
occurs cooperatively. The amount of eIF4A bound to an eIF4G fragment
containing both sites was greater than the sum of eIF4A bound to the
individual sites, the effect being less pronounced as both sites became
saturated (Fig. 5). Thus, the presence of one molecule of eIF4A on
eIF4G may accelerate or stabilize binding of the other. This model is
consistent with a recent observation that alteration of either one of
the eIF4A-binding sites by site-directed mutagenesis results in a form
of eIF4G that fails to bind to eIF4A, despite the presence of an intact
second site (28). The proposed cooperativity between sites may also
explain why eIF4F complexes purified from plant (31, 47, 48), yeast
(33), and Drosophila (35) do not contain eIF4A, despite the
fact that direct binding of eIF4A to eIF4G can be demonstrated in wheat
germ (49, 50) and yeast (51, 52) and that the affinity of yeast eIF4G
for eIF4A (Kd Recently the central domain of eIF4G was postulated to work as a
"ribosome recruitment core," in concert with other factors, based
on its ability to form 48 S pre-initiation complex and drive initiation
of translation (17, 27, 28). The COOH-terminal domain was proposed to
play a modulatory role in this process, since an eIF4G fragment
containing both eIF4A-binding sites is more active than one in which
the COOH-terminal site has been inactivated by site-directed
mutagenesis (28). Similarly, UV-cross-linking of RNA to eIF4A is
stimulated more by an eIF4G fragment containing both binding sites than
by either one separately (17). Our observation that binding of eIF4A to
eIF4G is cooperative could explain these results by ensuring a higher
affinity interaction between eIF4G and eIF4A.
Two models of interaction of mammalian eIF4G with eIF4A have been
proposed (28). One model would require that eIF4A have two different
surfaces for binding to eIF4G, allowing eIF4A to be sandwiched between
the central and COOH-terminal binding sites. It was suggested that a
resultant change in eIF4G conformation makes it more active in
translation. The other model proposes that the central domain of eIF4G
is sterically hidden from free eIF4A by the COOH-terminal domain. In
this model, eIF4A first binds to the COOH terminus and is later
transferred to the central region (28). Both models propose a 1:1
stoichiometry of binding eIF4A and eIF4G. We show here that eIF4G
fragments containing the individual eIF4A-binding sites compete with
each other and fail to form a sandwich on eIF4A. This indicates that
the determinants on eIF4A that are recognized by these two different
binding sites are either the same or overlapping, which is
incompatible with the first model. Titration experiments showed a 1:2
stoichiometry of binding of eIF4G to eIF4A, which is incompatible with
the second model. Also, the kinetic experiments indicate that eIF4A
binds faster to the central site than to the COOH-terminal site,
contrary to the second model. Thus, our data allow us to propose a new model whereby eIF4G binds two molecules of eIF4A simultaneously. Furthermore, binding of these two molecules of eIF4A to eIF4G appears
to occurs cooperatively. The implications of such a model on the
concerted action of paired eIF4A molecules acting to accomplish the
processive unwinding of mRNA remain to be explored.
1 s
1,
2.1 × 10
3 s
1,
and 17 nM for the central site and 5.1 × 103 M
1
s
1, 1.7 × 10
3
s
1, and 330 nM for the
COOH-terminal site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate on the bound
nucleotide has been shown to mediate changes in eIF4A conformation and
RNA affinity. ATP binding and hydrolysis produce conformational changes
in eIF4A that alter the RNA-protein interactions (8, 9, 3). A current
model for protein synthesis initiation envisions eIF4A in the role of
unwinding mRNA secondary structure in the 5'-untranslated region to
allow the 40 S ribosomal subunit to bind the mRNA and/or scan it
for the first AUG. The observation that dominant negative variants of
eIF4A inhibit translation is consistent with such a role (10).
Interestingly, the RNA helicase activity of eIF4A is ~20 times
greater when bound to eIF4G than as a free protein (6, 11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactoside was obtained from Indofine
Chemical Company (Belle Mead, NJ).
)4GT7EV (40), yielding the
plasmid pSK(
)4GT7SI. The sequence from BamHI (nt 1938 in eIF4G cDNA) to NotI (polylinker) were removed from
pSK(
)4GT7SI and inserted into plasmid pET-32a(+) at the
BglII and NotI sites. pTS4G(1078-1560) was
constructed by deleting a fragment from SpeI (polylinker) to
SpeI (nt 3334 in eIF4G cDNA) from pSK-HFCl (39), religating the larger restriction fragment, and inserting a restriction fragment from NotI (polylinker) to XhoI
(polylinker) derived from it between the NotI and
XhoI sites of pET-32a(+). pTS4G(877-1078) was constructed
by amplifying a sequence from pTS4G(613-1078) by 20 cycles of
polymerase chain reaction using the primers
5'-AGCTCCATGGTAATGCATGACTGTGTGGTC-3' and 5'-CCGCTGAGCAATAACTAG-3', the
latter being the T7 terminator primer 69337-1 from Novagen. The
products were digested with NcoI and XhoI and
inserted between the NcoI and XhoI sites of the
pET-32a(+) plasmid. pTS4G(975-1078) was constructed the same way,
except the primer 5'-AGCTCCATGGATAGACATCGAGAGCAC-3' was used together with the T7 terminator primer. pTS4G(1317-1560) was constructed by
inserting a restriction fragment from the NcoI (nt 3845 in eIF4G cDNA) to XhoI (polylinker) sites of
pCITE4Gwt (40) into pET-32a(+) between the NcoI
and XhoI sites. The eIF4G sequences in these plasmid were
verified by DNA sequencing at the Iowa State University facility.
-D-thiogalactoside
to a final concentration of 0.5-1 mM. Five hours after
induction at 30 °C, cells were harvested and frozen in liquid
N2. Thawed cell pellets were resuspended in lysis buffer
containing CompleteTM protease inhibitor tablets (Roche Molecular
Biochemicals) and disrupted by sonication. Cleared extracts were
incubated with nickel nitrilotriacetic acid-agarose (0.5 ml) with
rotation for 2 h at 4 °C. After several washes with buffer A
(50 mM Tris-HCl, 300 mM NaCl, 5 mM
-mercaptoethanol, 10% (v/v) glycerol, and 20 mM
imidazole), the recombinant proteins were eluted with buffer A
containing 200 mM imidazole.
-mercaptoethanol, 0.1% (v/v)
Tween 20, and 5% (v/v) glycerol, pH 7.5) to reduce the salt
concentration and then applied to a heparin-Sepharose CL-6B column
equilibrated with the same buffer except containing 100 mM
KCl (buffer B100). The protein was eluted with buffer
B400.
-mercaptoethanol, 0.1% (v/v) Tween 20, and 2 mM EDTA, pH 7.5) plus 5% (v/v) glycerol. Before SPR
analysis, purified eIF4G fragments and eIF4A were passed over the same
columns except equilibrated with buffer D (20 mM HEPES-KOH,
150 mM KCl, 2 mM EDTA, 0.05% Tween 20, and 0.5 mM
-mercaptoethanol, pH 7.5). After buffer exchange, the
concentrations of recombinant proteins were determined with the Bio-Rad
protein assay kit using bovine serum albumin as standard.
where BR is the binding ratio, i.e. the molar ratio
of bound eIF4A to the eIF4G fragment, n is number of
eIF4A-binding sites on the eIF4G fragment, [eIF4A]f is the
concentration of eIF4A not bound to the resin, and
Kd is the dissociation equilibrium constant for the
eIF4A·eIF4G complex. [eIF4A]f was calculated as the
difference between total eIF4A and bound eIF4A. A nonlinear least
squares fit was performed in which n and
Kd were allowed to vary.
(Eq. 1)
where n1 and
Kd1 represent the number of binding
sites and dissociation equilibrium constant for the first binding site,
respectively, and n2 and
Kd2 represent the number of binding
sites and dissociation equilibrium constant for the second.
(Eq. 2)
-mercaptoethanol, pH 7.5)
at a flow rate of 70 µl/min and contact time of 2 min, followed by
buffer D for 1 min.
AB). For this model, the function that describes the response (Rt) as a function of time
(t) during the injection phase is,
where Req is the response at equilibrium,
C is the molar concentration of S-eIF4G fragment,
t0 is time at the start of injection, and
ka and kd are association and
dissociation rate constants, respectively. For the post-injection phase, the response is,
(Eq. 3)
where R0 and t0
are the values of Rt and t, respectively, when
sample is replaced with buffer, and kd is the
dissociation rate constant. Values for the statistical closeness of
fit,
(Eq. 4)
2, were always below 1, indicating that the simple
1:1 model of interaction correctly described the experimental data.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of recombinant eIF4G
fragments used in this study. The aa residues present in each
fragment are indicated in parentheses. Binding sites for
other proteins are shown by the shaded and
cross-hatched boxes. The previous studies on which this
model is based have suggested that the eIF4A- and eIF3-binding sites in
the central domain are partially overlapping. The site of cleavage by
entero- and rhinovirus 2A proteases is shown by the arrow.
Molecular masses of eIF4G fragments plus the sequence tags are shown to
the right of each protein. PABP, poly(A)-binding
protein.
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Fig. 2.
Binding of eIF4A to eIF4G fragments.
[14C]eIF4A (0.6 µM) was incubated with
S-eIF4G(613-1078) (0.7 µM), S-eIF4G(1078-1560) (0.5 µM), S-eIF4G(1317-1560) (3.7 µM),
S-eIF4G(877-1078) (2.6 µM), S-eIF4G(975-1078) (2 µM), and S-protein-agarose in a volume of 30 µl.
Material bound specifically to the resin was eluted and subjected to
SDS-PAGE on 8% gels. A, Coomassie Blue staining.
B, autoradiography.
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Fig. 3.
eIF3 and eIF4A do not compete for binding to
eIF4G(613-1078). S-eIF4G(613-1078) was incubated at a
concentration of 0.09 µM with a 5-fold molar ratio of
[14C]eIF4A and a 2-, 5-, or 10-fold molar ratio of eIF3
(lanes 2, 3, and 4, respectively). The
same concentration of S-eIF4G(613-1078) was incubated with a 5-fold
molar ratio of eIF3 and a 2-, 5-, and 10-fold molar ratio of
[14C]eIF4A (lanes 5, 6, and
7, respectively). Controls included S-eIF4G(613-1078)
incubated in the absence of factors (lane 1) and
[14C]eIF4A and eIF3 incubated in the absence of
S-eIF4G(613-1078) (lane 8). All samples were treated with a
quantity of S-protein-agarose (15 µl) such that S-Protein was in a
10-fold molar excess over S-peptide-tagged eIF4G. A,
Coomassie Blue staining. B, autoradiography.
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Fig. 4.
Saturation analysis of eIF4A binding to eIF4G
fragments. A, S-eIF4G(613-1078) was incubated at a
concentration of 0.3 µM with eIF4A at 0, 0.071, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.35, 0.6, and 1.15 µM
(lanes 1 11, respectively). The
S-eIF4G(613-1078) and bound eIF4A were captured with a 10-fold molar
excess of S-protein-agarose, subjected to SDS-PAGE on an 8% gel, and
visualized with Coomassie Blue and autoradiography. B, the
amounts of S-eIF4G(613-1078) and eIF4A in A were
quantitated by autoradiography and by scanning stained bands and
comparing the signals to standard curves of the two purified proteins
electrophoresed on the same gel. BR is the molar ratio of eIF4A to
S-eIF4G(613-1078), and [eIF4A]f is the total eIF4A
concentration minus the complexed eIF4A. The curve is a least squares
fitting of Equation 1 to the data, in which n and
Kd are allowed to vary. The curve shown corresponds
to n = 1.1 and Kd = 96 nM. The coefficient of determination
(R2) was 0.96. Inset, replot of the
data as BR/[eIF4A]f versus BR. C, same
as A except that S-eIF4G(1078-1560) was used at 0.6 µM, and the eIF4A concentrations were 0.3, 0.65, 1, 2.65, and 5.3 µM for lanes 1-5, respectively.
D, quantitation of the data in C by the same
method as described in B. The curve shown corresponds to
n = 1.1 and Kd = 660 nM.
The R2 value was 0.94. Inset, the
data of D are replotted as in B, inset.
E, analysis of binding of S-eIF4G(613-1560) to varying
concentrations of eIF4A. The protocol was the same as in A,
except that S-eIF4G(613-1560) was used at 0.075 µM; the
concentrations of eIF4A were 0, 0.14, 0.18, 0.27, 0.36, 0.75, and 1.23 µM in lanes 1-7, respectively; and detection
of S-eIF4G(613-1560) and eIF4A were by Coomassie blue staining.
F, standard curves of purified eIF4G(642-1560) and eIF4A
were run on a different part of the same gel and used to quantitate the
values in E. The data are plotted as in B, but
the line corresponds to Equation 2 with n1 = 1.1, n2 = 1.1, Kd1 = 96 nM, and
Kd2 = 660 nM.
Inset, the data of F are replotted as in
B, inset.
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Fig. 5.
Comparative binding of eIF4A to eIF4G
fragments containing one or two eIF4A-binding sites. eIF4A at the
indicated concentrations was incubated with S-eIF4G(1078-1560),
S-eIF4G(613-1078), or S-eIF4G(613-1560), each at 0.1 µM. The S-eIF4G fragments and bound eIF4A were captured
on S-protein-agarose, subjected to SDS-PAGE on a 10% gel, and
quantitated with both Coomassie Blue and Western blotting using
standard curves for each protein run on the same gel.
S-eIF4G(613-1560) predicted (open bars) is calculated
assuming that there is no cooperativity between sites and represents
the sum of the binding ratios for S-eIF4G(1078-1560) and
S-eIF4G(613-1078) at the same eIF4A concentration.
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Fig. 6.
Kinetic analysis of eIF4A binding to
S-eIF4G(613-1078) and S-eIF4G(1078-1560) by SPR. A,
buffer D containing either 0, 3, 10, 25, 50, or 100 nM
S-eIF4G(613-1078) was passed over eIF4A immobilized on a sensor chip
(successive curves from lowest to highest). At 280 s, the mobile
phase was changed back to buffer D alone. Thin traces correspond to a
theoretical fitting of the data with a simple 1:1 kinetic binding model
using the ka and kd values given
in D. A "global fit" was used in which the single best
values of ka and kd were obtained
for all concentrations of analyte. The sensor chip contained about 300 RU of immobilized eIF4A. B, the same as A except
that 0, 10, 50, 250, 450, 600, and 1000 nM solutions of
S-eIF4G(1078-1560) were passed over the sensor chip containing 250 RU
of eIF4A. C, S-eIF4G(975-1078), which does not contain an
eIF4A-binding site (see Fig. 2), was passed over a chip containing 200 RU eIF4A at concentrations of 0, 3, 10, 25, 50, 100, 200, and 300 nM. The response curves closest to the base line correspond
to the lowest concentration. D, association
(ka) and dissociation (kd) rate
constants calculated from the data in A and B
using a global fit of the 1:1 binding model. The dissociation
equilibrium constant Kd is calculated as
kd/ka. The 2 value
is a statistical measure of the closeness of fit. Residuals are the
maximal deviation between actual and theoretical responses.
2 (Fig. 6D) indicate that the 1:1 binding
model provides a good fit to the data. The rate constants obtained for
association (ka) and dissociation
(kd) are shown in Fig. 6D. An apparent dissociation constant, Kd, can be calculated from
kd/ka (Fig. 6D).
However, it should be noted that there may be hidden intermediates in
the binding of eIF4A to eIF4G, and the rate constants obtained only
reflect the slowest step in this process. The results show that
S-eIF4G(613-1078) associates with eIF4A about 24-fold faster than does
S-eIF4G(1078-1560). S-eIF4G(613-1078) also dissociates from eIF4A
1.2-fold faster than does S-eIF4G(1078-1560). As a result,
S-eIF4G(613-1078) has an affinity for eIF4A that is 19-fold higher
than S-eIF4G(1078-1560). Kd values obtained by SPR
are considerably lower than those obtained by S-protein-agarose for the
same protein-protein interactions, (17 versus 96 nM for S-eIF4G(613-1078) and 330 versus 660 nM for S-eIF4G(1078-1560)). This is consistent with the
fact that the S-protein-agarose protocol represents a nonequilibrium
condition and is expected to underestimate binding affinities.
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Fig. 7.
Competition between eIF4G(642-1078) and
S-eIF4G(1078-1560) for binding to eIF4A. eIF4G(642-1078), which
contains no S-tag, was produced by treatment of S-eIF4G(613-1078) with
Coxsackievirus 2A protease. S-eIF4G(1078-1560) (0.085 µM) was incubated with a 2-fold molar ratio of
[14C]eIF4A and either no eIF4G(642-1078) (lane
2) or a 2-, 5-, or 10-fold molar ratio of eIF4G(642-1078)
(lanes 3, 4, and 5, respectively).
Lane 6 is the same as lane 4 except that 0.05 µM eIF3 was also present. Lanes 7-9 are the
same as lanes 3-5 except that no S-eIF4G(1078-1560) was
present. Lane 10 represents binding of S-eIF4G(613-1078)
(the same molar concentration as eIF4G(642-1078) in lane 3)
with eIF4A. Lane 1 is a control with
[14C]eIF4A but no S-eIF4G(1078-1560). In all cases, the
S-eIF4G(1078-1560) and bound eIF4A were captured by incubation with a
10-fold molar excess of S-protein-agarose and subjected to SDS-PAGE on
a 10% gel. A, Coomassie Blue staining. B,
Western blotting using anti-eIF4G peptide 9 antibodies. C,
autoradiogram of [14C]eIF4A. Numbers under the
lanes represent the molar ratio of eIF4A to
S-eIF4G(1078-1560), expressed as a percentage of lane
2.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
s
1), but it also dissociates from
eIF4G(613-1078) 1.2-fold faster than from eIF4G(1078-1560)
(kd = 2.1 × 10
3
versus 1.7 × 10
3
s
1). This more rapid turnover of eIF4A bound
to the central site may have mechanistic consequences.
30 nM; Ref. 52) is
comparable with that of the central site of human eIF4G
(Kd = 17 nM; Fig. 6). These plant and
yeast eIF4Gs have high homology to the central domain of human eIF4G
but low homology to the COOH-terminal domain. Consequently, there may
be no enhancement of eIF4A binding by the COOH-terminal domain in the
nonmammalian eIF4Gs.
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ACKNOWLEDGEMENTS |
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We thank Dr. Hans Trachsel for providing anti-eIF4A antibodies and Aili Cai, Minghua Chen, and Clint Waddell for valuable technical assistance.
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FOOTNOTES |
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* This work was supported by NIGMS, National Institutes of Health Grants GM20818 and GM26796.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: ProdiGene, 101 Gateway Blvd., Suite 100, College Station, TX 77845.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932. Tel.: 318-675-5161; Fax: 318-675-5180; E-mail: rrhoad@lsuhsc.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M006345200
2 The nomenclature system recommended by an ad hoc committee appointed by the IUBMB is used here (53). However, since this work concerns only eIF4G-1, the term eIF4G will subsequently refer to eIF4G-1.
3 N. L. Korneeva and R. E. Rhoads, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: eIF, eukaryotic initiation factor; aa, amino acid residues; BR, molar binding ratio of eIF4A to eIF4G fragments; PAGE, polyacrylamide gel electrophoresis; RU, response unit; SPR, surface plasmon resonance; nt, nucleotide(s).
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