From the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Jack and Pearl Resnick Campus, Bronx, New York 10461
Received for publication, October 9, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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We have examined the role of the mammalian
initiation factor eIF1 in the formation of the 40 S preinitiation
complex using in vitro binding of initiator Met-tRNA (as
Met-tRNAi·eIF2·GTP ternary complex) to 40 S ribosomal
subunits in the absence of mRNA. We observed that, although both
eIF1A and eIF3 are essential to generate a stable 40 S preinitiation
complex, quantitative binding of the ternary complex to 40 S subunits
also required eIF1. The 40 S preinitiation complex contained, in
addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with
respect to the bound Met-tRNAi. These three initiation
factors also bind to free 40 S subunits, and the resulting complex can
act as an acceptor of the ternary complex to form the 40 S
preinitiation complex (40 S·eIF3·eIF1·eIF1A·Met-tRNAi·eIF2·GTP). The
stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well
as to the 40 S preinitiation complex was stabilized by the presence of
both eIF1 and eIF3. These studies suggest that it is possible for eIF1
and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.
The initiation of translation in eukaryotic cells occurs by a
sequence of partial reactions that require a number of specific proteins called eukaryotic (translation) initiation factors
(eIFs).1 According to the
currently accepted view of translation initiation, primarily derived
from in vitro studies with purified initiation factors, an
obligatory intermediate step in the overall initiation reaction is the
binding of the initiator Met-tRNAi as the
Met-tRNAi·eIF2·GTP ternary complex to a 40 S ribosomal
subunit containing bound initiation factor eIF3. This interaction leads
to the production of the 40 S preinitiation complex (40 S·eIF3·Met-tRNAi·eIF2·GTP). The 40 S preinitiation
complex then binds to the 5'-capped end of mRNA and scans the
mRNA in a 5' In addition to the initiation factors described above, the 17-kDa eIF1A
and the 12-kDa eIF1 are also known to play essential roles in the
overall initiation process (1-5). Earlier biochemical studies
demonstrated that both eIF1 and eIF1A have a weak stimulatory effect on
the binding of Met-tRNAi and mRNA to 40 S and 80 S
initiation complexes in the presence of other factors (6-11). The
presence of eIF1A in the 40 S initiation complex was also shown in one of these earlier studies (9). In vivo studies in
Saccharomyces cerevisiae demonstrated that the genes
encoding these two small initiation factors are essential for
initiation of protein synthesis and required for cell growth and
viability (12-14). These observations are in accord with genetic
studies in S. cerevisiae (15, 16) that indicate that eIF1
(SUI 1) plays an essential role in the fidelity of start
site selection. In a purified mammalian translation initiation system,
Pestova et al. (17), using toe-printing analysis on natural
Recently we described (19, 20) an in vitro translation
initiation assay that specifically measures the transfer of
Met-tRNAi (as Met-tRNAi·eIF2·GTP ternary
complex) to 40 S ribosomal subunits in the absence of mRNA or an
AUG codon to form the 40 S preinitiation complex. Using this assay, we
observed that both eIF1A and eIF3 were essential for the efficient
transfer of Met-tRNAi·eIF2·GTP ternary complex to 40 S
ribosomal subunits to form a stable 40 S preinitiation complex (20).
However, analysis of the 40 S preinitiation complex showed that,
although eIF3 was bound to the complex, eIF1A was not. It was of
interest, therefore, to examine whether eIF1 has any role in the
formation of the preinitiation complex as well as in the stable
association of eIF1A to this complex.
In the present work, we have used a modified initiation assay to show
that, although eIF1, by itself, does not stimulate the formation of the
40 S preinitiation complex, at low concentrations of initiation
factors, all three factors eIF1, eIF1A, and eIF3 are required for the
quantitative binding of the Met-tRNAi·eIF2·GTP ternary
complex to 40 S ribosomal subunits to form the stable 40 S
preinitiation complex. Furthermore, analysis of the in vitro assembled 40 S preinitiation complex shows that both eIF1 and eIF1A are
present in a near 1:1 stoichiometry with respect to bound
Met-tRNAi. We also demonstrate that these three initiation factors bind to 40 S ribosomal subunits in the absence of
Met-tRNAi·eIF2·GTP ternary complex and the resulting 40 S·eIF3·eIF1·eIF1A can then bind the
Met-tRNAi·eIF2·GTP ternary complex to form the 40 S
preinitiation complex (40 S·eIF3·eIF1·eIF1A·Met-tRNAi·eIF2·GTP). The
implications of these results in relation to 40 S preinitiation complex
formation are discussed.
tRNA, Ribosomal Subunits, Purified Proteins, and
Antibodies--
The preparation of 35S- or
3H-labeled rabbit liver initiator Met-tRNAi
(10,000 and 66,000 cpm/pmol, respectively) and 40 S and 60 S ribosomal
subunits from Artemia salina eggs were as described previously (21). Purified eIF2 and eIF3 from rabbit reticulocyte lysates and bacterially expressed recombinant human eIF1A protein (unlabeled or labeled with 35S) were isolated as described
elsewhere (19). Rabbit IgG antibodies specific for mammalian eIF1A were
obtained as described previously (19), and total IgY antibodies
specific for mammalian eIF3 subunits were isolated from egg yolks of
laying hens immunized with purified rabbit reticulocyte eIF3 (22).
Polyclonal antibodies against purified denatured eIF1 were prepared in
rabbits following a procedure similar to that used previously for
preparation of anti-eIF1A antibodies (19). Immunoblot analysis was as
described previously (23). The mixture of protease inhibitors added to
buffer solutions used during purification of recombinant proteins from
bacterial cell extracts consisted of leupeptin (0.5 µg/ml), pepstatin
A (0.7 µg/ml), aprotinin (2 µg/ml), and freshly prepared
phenylmethylsulfonyl fluoride (1 mM).
Expression of eIF1 in Escherichia coli and Purification of the
Recombinant Protein--
The open reading frame of eIF1 cDNA (24)
was synthesized by reverse transcription-polymerase chain reaction of
HeLa poly(A)+ RNA using the Invitrogen kit and appropriate
oligonucleotide primers corresponding to the N-terminal and C-terminal
ends of the eIF1 open reading frame. These primers had
NdeI/EcoR1 overhangs. The PCR product was
sequenced to ensure error-free DNA synthesis and cloned into the
NdeI/EcoRI sites of pET-5a plasmid (Novagen). This pET-5a-eIF1 expression vector was used to transform E. coli BL21(DE3) cells (Novagen). Transformants were then grown at
37 °C in 1 liter of LB medium (25) containing 50 µg/ml ampicillin to an A600 of about 1.2, induced with 1 mM isopropyl-
For purification of recombinant eIF1, frozen E. coli cells
(5 g) were disrupted by sonication in a buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2,
50 mM KCl, and 1 mM EDTA, and the postribosomal
supernatant was prepared as described previously for the isolation of
recombinant eIF5 from overproducing E. coli cells (26). The
postribosomal supernatant (170 mg of protein in 16 ml of total volume)
was loaded onto a 60-ml bed volume of DEAE-cellulose column
equilibrated in buffer A (20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, and 10%
glycerol) plus 50 mM KCl, and the column was washed with
buffer A plus 50 mM KCl. eIF1 was virtually unretarded
under these conditions and appeared in the initial wash along with the
unretarded protein peak. Fractions containing the protein peak were
pooled (26 mg of protein), adjusted to 0.1 M KCl and loaded
onto a 9-ml bed volume of a phosphocellulose column equilibrated in
buffer A plus 100 mM KCl. The column was washed with the
same buffer until A280 of the effluent was below
0.1. Bound eIF1 was then eluted from the column with a linear gradient
(total volume, 72 ml) in buffer A from 100 mM KCl to 800 mM KCl. Fractions containing eIF1 (eluting at about 450 mM KCl) were pooled, dialyzed against buffer A plus 50 mM KCl to reduce the ionic strength to that of buffer A
plus 100 mM KCl, and then applied to a 1-ml bed volume
fast-protein liquid chromatography Mono Q column. The column was washed
with 5 ml of buffer A plus 100 mM KCl, and bound proteins
were eluted by a gradient elution (12.5 ml of total volume) from buffer
A plus 100 mM KCl to buffer A plus 400 mM KCl.
Fractions containing eIF1 (eluting at about 150 mM KCl)
were pooled, dialyzed against buffer B (20 mM Tris-HCl, pH
7.5, 2.5 mM 2-mercaptoethanol, 100 mM KCl, 0.1 mM EDTA, and 50% glycerol), and stored at Preparation of 3H-Labeled eIF1--
E.
coli BL21(DE3) harboring the pET-5a-eIF1 expression plasmid was
grown at 37 °C in 500 ml of M9 medium (25) containing 50 µg/ml
ampicillin to A600 of about 1.0, induced with 1 mM isopropyl- Assay for Formation of the 40 S Preinitiation Complex--
40 S
preinitiation complex formation was measured by the binding of
35S- or 3H-labeled Met-tRNAi (as
the Met-tRNAi·eIF2·GTP ternary complex) to 40 S
ribosomal subunits at 1 mM Mg2+ in the absence
of mRNA or AUG as follows. Reactions were carried out in two
stages. In Stage 1, reaction mixtures (100 µl each) containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM 2-mercaptoethanol, 4 µg of nuclease-free bovine serum
albumin, 400 µM GTP, 1.2 µg of purified rabbit
reticulocyte eIF2, and 8 pmol of either
[35S]Met-tRNAi (30,000-60,000 cpm/pmol) or
[3H]Met-tRNAi (10,000 cpm/pmol) were
incubated at 37 °C for 4 min to promote the formation of the
[35S]Met-tRNAi·eIF2·GTP or the
[3H]Met-tRNAi·eIF2·GTP ternary
complex. Reaction mixtures were then chilled in an ice-water bath, and
a 5-µl aliquot of the reaction mixture was subjected to
nitrocellulose membrane filtration to determine the amount of the
ternary complex formed (27). In Stage 2, another set of reaction
mixtures (50 µl each) containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM MgCl2, 2.5 mM 2-mercaptoethanol (Buffer B), 0.6 A260 units of 40 S ribosomal subunits and, where indicated, 1.5-15 µg of eIF3, 50 ng to 1 µg of eIF1A, and 50-500 ng of eIF1 were incubated at 37 °C for 4 min and then supplemented with 95 µl of the Stage 1 reaction mixture containing about 4 pmol of
35S- or 3H-labeled
Met-tRNAi·eIF2·GTP ternary complex. The
Mg2+ concentration of each reaction mixture (now 175 µl
each) was adjusted to 1 mM, and the reaction mixtures were
incubated at 37 °C for 4 min, chilled in an ice-water bath, and then
layered onto a 5-ml 7.5-30% (w/v) sucrose density gradient containing buffer B and centrifuged at 48,000 rpm for 105 min in a SW 50.1 rotor.
Fractions (200-300 µl) were collected from the bottom of each
gradient, and the radioactivity in each fraction was determined in a
liquid scintillation spectrometer. The efficiency of the 40 S
preinitiation complex formation was calculated relative to 35S- or 3H-labeled
Met-tRNAi·eIF2·GTP ternary complex.
Assay for the Association of eIF1A and eIF1 with the 40 S
Preinitiation Complex--
The 40 S preinitiation complex
containing [3H]- or [35S]methionine-labeled
Met-tRNAi was formed as described above under "Assay for
Formation of the 40 S Preinitiation Complex," except that either
[35S]eIF1A (1900 cpm/pmol) or [3H]eIF1 (820 cpm/pmol) replaced unlabeled eIF1A or eIF1, respectively. In reaction
mixtures containing [35S]eIF1A,
[3H]Met-tRNAi was used for the ternary
complex formation in Stage 1 and unlabeled eIF3 and eIF1 were used in
Stage 2 reactions. In reactions containing [3H]eIF1,
[35S]Met-tRNAi was used for ternary complex
formation in Stage 1 and unlabeled eIF3 and eIF1A were used in Stage 2. Following incubation to form the 40 S preinitiation complex, the
reactions were analyzed either by sucrose gradient centrifugation as
described above or by Sephadex G-75 gel filtration. For analysis by
sucrose gradient centrifugation, 0.2- to 0.3-ml fractions were
collected from the bottom of each tube following centrifugation, and
the radioactivity in each fraction was measured by liquid scintillation
counting. The presence of eIF3 in each fraction was determined by
Western blotting.
For analysis by Sephadex G-75 gel filtrations, chilled reaction
mixtures were each applied to a 12-ml bed volume column of Sephadex
G-75 previously equilibrated in 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol, and 5% glycerol. The column was then
developed with the same equilibrating buffer at 4 °C. Fractions of
250 µl were collected and assayed for 3H and/or
35S radioactivity by counting each fraction in Aquasol in a
liquid scintillation spectrometer. The elution positions of the 40 S preinitiation complex, free eIF1A, eIF1, or eIF3 proteins, and free
Met-tRNAi were determined separately in the same column.
Role of eIF1 in the Formation of the 40 S Preinitiation
Complex--
We have previously used an in vitro assay that
measures the binding of Met-tRNAi (as the
Met-tRNAi·eIF2·GTP ternary complex) to 40 S ribosomal
subunits in the absence of mRNA or AUG codon to form the 40 S
preinitiation complex (19, 20). Although these initiation assays were
carried out in reactions containing 1 mM Mg2+,
they were subsequently analyzed for the binding of
Met-tRNAi to 40 S ribosomes employing sucrose gradients in
buffers containing 5 mM Mg2+. Elevated levels
of Mg2+ are known to stabilize ribosomal binding of
Met-tRNAi and have been used by all investigators to
analyze 40 S and 80 S initiation complexes in vitro by
sucrose gradient centrifugation (7-11, 19, 20). Using such an assay,
we showed that two initiation factors, eIF1A and eIF3, are required for
efficient formation of a stable 40 S preinitiation complex. Although
eIF1A promotes the transfer of Met-tRNAi·eIF2·GTP
ternary complex to 40 S ribosomal subunits to form the 40 S
preinitiation complex, the presence of eIF3 bound to the 40 S ribosomal
subunits is required to stabilize the resulting complex under a variety
of experimental conditions (20).
To determine if eIF1 has a role in the formation and/or stability of
the 40 S preinitiation complex, we used a similar assay system (19, 20)
except that both the initiation reactions and the subsequent sucrose
gradient centrifugation analysis were carried out in buffers containing
1 mM Mg2+. We reasoned that the presence of 5 mM Mg2+ in sucrose gradient buffers might
artificially stabilize the bound Met-tRNAi and thus prevent
the identification of additional factors involved in the 40 S
preinitiation complex formation. For this reason a preformed
[35S]Met-tRNAi·eIF2·GTP ternary complex
(3.5 pmol) was incubated, at physiological 1 mM
Mg2+ with 40 S ribosomal subunits and saturating levels of
each of the three initiation factors, eIF1A, or eIF1, or eIF3, either alone or in combination. The products were analyzed by sucrose gradient
centrifugation in buffers containing 1 mM Mg2+
(Fig. 1). When tested alone, eIF1A was
the most effective of the three initiation factors in supporting
binding of Met-tRNAi to 40 S ribosomal subunits. Although
eIF1A, by itself, promoted the transfer of about 1.6 pmol of
Met-tRNAi, eIF3 or eIF1, when used alone, supported binding
of only 0.6 and 0.35 pmol of Met-tRNAi, respectively, to 40 S ribosomes. When both eIF1A and eIF3 were present in the preinitiation
reaction, the binding of Met-tRNAi to 40 S ribosomal
subunits was increased to about 2.4 pmol. Surprisingly, although eIF1,
by itself, did not promote significant 40 S preinitiation complex
formation, its presence in the eIF1A-mediated reaction caused a marked
stimulation of preinitiation complex production. About 2.9 pmol of the
Met-tRNAi was bound to 40 S ribosomes under these
conditions accounting for >80% of the input
Met-tRNAi·eIF2·GTP ternary complex (Fig. 1). In the
presence of all three initiation factors, eIF1A, eIF1, and eIF3,
Met-tRNAi was quantitatively transferred to the 40 S
particle to form the 40 S preinitiation complex (Fig. 1). Omission of
eIF1A from such a reaction markedly decreased the binding of
Met-tRNAi to 40 S ribosomal subunits (Fig. 1). These
results suggest that, although eIF1A is primarily responsible for
transferring Met-tRNAi (as
Met-tRNAi·eIF2·GTP ternary complex) to 40 S ribosomes,
the efficiency of this reaction is maximal in the presence of all three
initiation factors. It should be noted that, in the experiments
presented in Fig. 1, the binding of Met-tRNAi (present as
[35S]Met-tRNAi·eIF2·GTP ternary complex)
to 40 S ribosomal subunits was monitored. To demonstrate that this
binding corresponds to the entire ternary complex, we carried out
these reactions with [3H]Met-tRNAi·eIF2·[
The above results were obtained using saturating levels of each of the
three initiation factors. However, in vivo, these factors are presumably present in much lower concentrations. The stimulatory effect of eIF1 on eIF1A-promoted transfer of Met-tRNAi to
40 S ribosomes became more pronounced when 40 S preinitiation complex formation was measured in the absence of eIF3 as a function of eIF1 and
eIF1A concentrations. As shown in Fig. 2,
lower concentrations of either eIF1A or eIF1, when used alone,
supported the 40 S preinitiation complex production inefficiently.
However, the presence of both eIF1A and eIF1 in preinitiation reactions
caused a marked increase in the level of the 40 S preinitiation
complex.
The reactions described above were performed with isolated 40 S
ribosomal subunits. However, in vivo, both 40 S and 60 S
ribosomal subunits are present in the same milieu. Thus, to investigate stable 40 S preinitiation complex formation under more physiological conditions, i.e. in the presence of both 40 S and 60 S
ribosomal subunits, a preformed
[35S]Met-tRNAi·eIF2·GTP ternary complex
was incubated with 40 S ribosomal subunits and various combinations of
eIF1A, eIF1, and eIF3 to form the 40 S preinitiation complex. The
products were then treated with 60 S ribosomal subunits after raising
the Mg2+ concentration to 5 mM, conditions that
favor spontaneous association of the ribosomal subunits. Analysis by
sucrose gradient centrifugation revealed that the 40 S preinitiation
complex formed with either eIF1A or eIF1 was disrupted nearly
completely by 60 S ribosomal subunits, whereas the preinitiation
complex formed with eIF3 alone was moderately stable (Fig.
3). Furthermore, although eIF1A and eIF1
together promoted significant 40 S preinitiation complex formation,
nearly 60% of the resulting complex was not stable in the presence of
60 S ribosomal subunits (Fig. 3). In contrast and in keeping with
previous results from this laboratory (20), when eIF1A and eIF3 were
used to form the 40 S preinitiation complex, ~70% of the complex was
stable in the presence of 60 S ribosomal subunits. However, when all
three initiation factors were used to form the 40 S preinitiation
complex, 60 S ribosomal subunits failed to destabilize the complex
formed and nearly 100% of the preinitiation complex was stable (Fig.
3).
Fate of eIF1 and eIF1A during Formation of the 40 S Preinitiation
Complex--
To determine the fate of eIF1 and eIF1A during
factor-dependent formation of the 40 S preinitiation
complex, we purified recombinant 3H-labeled eIF1 and
35S-labeled eIF1A ("Experimental Procedures").
Experiments were carried out in which a 40 S preinitiation complex was
formed by incubating
[35S]Met-tRNAi·eIF2·GTP ternary complex
with 40 S ribosomal subunits and [3H]eIF1 in the presence
of unlabeled eIF1A and eIF3. In a separate experiment, another 40 S
preinitiation complex was formed by incubating the
[3H]Met-tRNAi·eIF2·GTP ternary complex
with 40 S ribosomal subunits and [35S]eIF1A in the
presence of unlabeled eIF1 and eIF3. Each reaction product was then
subjected to sucrose gradient centrifugation. As shown in Fig.
4, a fraction of labeled eIF1
(panel A) and eIF1A (panel B) co-sedimented with
Met-tRNAi bound to the 40 S particle. Based on the level of
radioactive eIF1 and eIF1A bound to the 40 S preinitiation complex,
each of these factors was present in the 40 S preinitiation complex in
a stoichiometry of ~1:1 with respect to the bound initiator Met-tRNA
(Fig. 4). Western blot analysis using anti-eIF3 antibodies also
detected eIF3 in the preinitiation complex in agreement with previous
results (20) (Fig. 4C). These findings indicate that all
three initiation factors, eIF3, eIF1A, and eIF1, can associate with the
40 S preinitiation complex.
The requirements for the binding of eIF1 and eIF1A to the 40 S
preinitiation complex were also examined using a similar assay (Fig.
5). The binding of eIF1 was dependent on
the simultaneous presence of eIF3. Although omission of eIF1A resulted
in a moderate decrease in the binding of eIF1 relative to
Met-tRNAi bound in the 40 S preinitiation complex, in the
absence of eIF3 the amount of bound eIF1 was drastically reduced (Fig.
5). In contrast, eIF1A, in the presence of eIF1, can bind the 40 S
preinitiation complex in the absence of eIF3, although the efficiency
of binding was greatly reduced as compared with the binding in the
presence of eIF3. Additionally, omission of eIF1 led to a near-complete
abolition of eIF1A binding to the 40 S ribosomes. These findings agree
with our previous results (19, 20), showing that, when the 40 S preinitiation complex was formed in reactions containing eIF1A and eIF3
but lacking eIF1, eIF1A did not associate with the 40 S preinitiation
complex.
In the experiments described above, it was somewhat surprising that,
although eIF1, in the absence of eIF3, stimulated the eIF1A-promoted
transfer of Met-tRNAi to 40 S ribosomal subunits, eIF1 was
not associated with the resulting 40 S complex. Likewise, in the
absence of eIF1, eIF1A was also not bound to the 40 S preinitiation complex. The possibility exists that the association of eIF1 and eIF1A
to the 40 S preinitiation complex in the absence of eIF3 or eIF1,
respectively, was much weaker than when all three factors were present.
Because the binding analysis (Fig. 5) was carried out by sucrose
gradient centrifugation, conditions known to exert considerable
hydrostatic pressure on the sedimenting particles, it was likely that
in the absence of eIF3 and eIF1, respectively, both eIF1 and eIF1A
dissociated from the 40 S complex during the sucrose gradient
centrifugation. To examine this possibility, binding analyses were also
performed using gel filtration. For this purpose, the 40 S
preinitiation complex was formed by incubating (a) the
[35S]Met-tRNAi·eIF2·GTP ternary complex
with 40 S ribosomes, [3H]eIF1, and unlabeled eIF1A and
eIF3 and (b) the
[3H]Met-tRNAi·eIF2·GTP ternary complex
with 40 S ribosomes, [35S]eIF1A, and unlabeled eIF1 and
eIF3. Each reaction product was then subjected to Sephadex G-75 gel
filtration (Fig. 6). In each case, as
expected, labeled Met-tRNAi bound to the 40 S ribosomes eluted from the column in the excluded volume (data not shown). In
agreement with the results obtained by sucrose gradient analysis (Fig.
5), both [3H]eIF1 and [35S]eIF1A were
detected in the excluded fraction (Fig. 6, A and D) consistent with their association with the 40 S
preinitiation complex. In the absence of 40 S ribosomal subunits,
neither radiolabeled Met-tRNAi (not shown) nor
[3H]eIF1 or [35S]eIF1A was detected in the
excluded material (Fig. 6, B and E). However,
when the preinitiation complex was formed in the absence of eIF3,
[3H]eIF1 was still detected in the excluded material,
although the amount of bound [3H]eIF1 was drastically
reduced in this case (Fig. 6, compare C with A).
Likewise, in reactions containing [35S]eIF1A, omission of
eIF1 did not abolish the binding of [35S]eIF1A. Rather,
the binding was somewhat reduced (Fig. 6, compare F with
D). In sucrose gradient centrifugation analysis (see Fig. 5), neither eIF1 nor eIF1A was associated with the 40 S preinitiation complex under these conditions. Taken together, these results show that
eIF1 and eIF1A were still associated with the 40 S preinitiation complex in the absence of either eIF3 or eIF1, respectively. However, their association was considerably weakened under these conditions, resulting in the dissociation of eIF1 and eIF1A from the 40 S preinitiation complex during sucrose gradient centrifugation (due to
the hydrostatic pressure exerted on the sedimenting particles during
the centrifugation).
Interaction of eIF1 and eIF1A with Free 40 S Ribosomal
Subunits--
Several laboratories have reported that mammalian eIF3
binds to 40 S ribosomal subunits in vitro in the absence of
all other initiation components and the resulting 40 S·eIF3 complex
is stable to sucrose gradient centrifugation (20, 28, 29). There is also evidence that, in vivo, the majority of native 40 S
ribosomal subunits contain bound eIF3 (30, 31). We examined whether both eIF1 and eIF1A can also associate with free 40 S ribosomal subunits in the absence of the Met-tRNAi·eIF2·GTP
ternary complex. Experiments were carried out in which free 40 S
ribosomal subunits were incubated with either [3H]eIF1
and unlabeled eIF1A and eIF3, or with [35S]eIF1A and
unlabeled eIF1 and eIF3, in the absence of
Met-tRNAi·eIF2·GTP ternary complex, and the products
were analyzed by Sephadex G-75 gel filtration. Under these conditions,
both [3H]eIF1 and [35S]eIF1A were detected
in the excluded fractions (Fig. 7,
A and F). In the absence of 40 S ribosomal
subunits, neither 35S nor 3H radioactivity was
eluted in the excluded fractions (Fig. 7, B and
G). These results suggest that both eIF1 and eIF1A bind to
40 S particles in the absence of bound
Met-tRNAi·eIF2·GTP ternary complex. Analysis of the
requirements for the binding of eIF1 to 40 S ribosomal subunits shows
that, in the absence of eIF3, binding of [3H]eIF1 was
abolished almost completely (Fig. 7D). In contrast, in
reactions containing eIF3 but no eIF1A, there was still significant but
reduced binding of [3H]eIF1 to 40 S ribosomes (Fig.
7C). No detectable binding of [3H]eIF1 to 40 S
ribosomes was observed in the absence of both eIF1A and eIF3 (Fig.
7E).
The requirements for eIF1A binding to 40 S ribosomal subunits were also
examined. In this case, the binding of [35S]eIF1A was
only somewhat reduced in the absence of either eIF1 (Fig.
7H) or eIF3 (Fig. 7I). Even in the absence of
both eIF1 and eIF3, low but detectable levels of eIF1A were still bound to the 40 S ribosomal subunits (Fig. 7J). Taken together,
these results suggest that the binding of eIF1 to 40 S ribosomal
subunits requires the presence of eIF3 and that, although eIF1A alone
can bind to 40 S ribosomal subunits, this binding is stimulated
markedly in the presence of both eIF1 and eIF3. It should be noted that when the binding analyses presented in Fig. 7 (A and
F) were carried out by sucrose gradient centrifugation, no
binding of either [3H]eIF1 or [35S]eIF1A
was observed (data not shown). These results indicate that the
association of eIF1 and eIF1A with free 40 S ribosomal subunits is
relatively weak. This is also reflected by the trailing of the
radioactivity of eIF1 and eIF1A in the elution profiles from gel
filtrations presented in Fig. 7.
To investigate whether preformed 40 S·eIF3·eIF1·eIF1A complex can
serve as an acceptor for the Met-tRNAi·eIF2·GTP ternary complex, we isolated 40 S·eIF3·eIF1·eIF1A free of unreacted
reaction components by Sephadex G-75 gel filtration. Incubation of this isolated complex with Met-tRNAi·eIF2·GTP resulted in
the formation of the 40 S preinitiation complex (Fig.
8). Furthermore, all three initiation
factors, eIF1, eIF1A, and eIF3, remained bound to the 40 S
preinitiation complex (Fig. 8).
In the initiation of protein synthesis, the binding of the
initiator Met-tRNA (as Met-tRNAi·eIF2·GTP ternary
complex) to 40 S ribosomal subunits is an obligatory intermediate step
in the selection of a start codon in mRNA. This binding, which
occurs in the absence of mRNA, leads to the formation of the 40 S
preinitiation complex.
In studies presented here, we have investigated the initiation factor
requirements for formation of a stable 40 S preinitiation complex using
translation initiation assays that differed from those used previously
(20) in that, following initiation reactions at 1 mM
Mg2+, subsequent analyses of 40 S preinitiation complex
formation were performed at 1 mM Mg2+ rather
than 5 mM Mg2+. We show that, although eIF1A
and eIF3 together (in the absence of eIF1) promoted efficient formation
of the stable 40 S preinitiation complex, the transfer of
Met-tRNAi (as Met-tRNAi·eIF2·GTP ternary complex) was not quantitative. Quantitative transfer of
Met-tRNAi was only observed when eIF1 was also present.
Under the modified conditions described here, all three factors were
associated with the 40 S preinitiation complex. Furthermore, the 40 S
preinitiation complex formed in the presence of all three factors was
completely stable even in the presence of 60 S ribosomal subunits. It
appears that, at 1 mM Mg2+, eIF1 plus eIF1A
promotes the transfer of Met-tRNAi·eIF2·GTP ternary
complex to the 40 S ribosomal subunit, and this effect of eIF1 becomes
more evident when lower concentrations of the factors are used in the
assays. At these lower concentrations, although eIF1A and eIF1 by
themselves cannot promote transfer of Met-tRNAi to 40 S
ribosomal subunits, the presence of both these factors leads to a
marked stimulation in 40 S complex formation. However, in the absence
of eIF3, eIF1 was not detected in the resulting 40 S preinitiation
complex (analyzed by sucrose gradient centrifugation). These
observations indicated that, although eIF1 can carry out its function
in the absence of eIF3, the presence of eIF3 is required for the stable
association of eIF1 with the 40 S preinitiation complex. These results
are in agreement with the observation (14, 32) that, in S. cerevisiae, eIF3 and eIF1 interact and are present in a
multifactor complex (33). Similar interaction between mammalian eIF1
and eIF3 has also been observed in glutathione S-transferase
pull-down assays (34). In contrast to eIF1, the stable eIF1A binding to
the 40 S preinitiation complex, measured by sucrose gradient
centrifugation, was not observed when the complex was formed in the
presence of eIF1A and eIF3. Rather, binding of eIF1A to the 40 S
preinitiation complex was dependent on the presence of eIF1. This
observation explains our previous results (19, 20) that, although
eIF1A, by itself or in the presence of eIF3, can promote significant 40 S preinitiation complex formation, the resulting 40 S complex did not
contain eIF1A, because these assays were carried out in the absence of eIF1.
The results presented in this report show that eIF3, eIF1, and eIF1A
can also bind to free 40 S ribosomal subunits to form the 40 S·eIF3·eIF1·eIF1A complex (Fig. 7). Here again, the binding of
eIF1 to free 40 S subunits appears to be dependent on the presence of
eIF3. Likewise, although eIF1A, by itself, can bind weakly to 40 S
ribosomal subunits, maximal binding of eIF1A requires the presence of
both eIF1 and eIF3. Finally, it is important to note that the 40 S·eIF3·eIF1·eIF1A complex, itself, can act as an acceptor of the
Met-tRNAi·eIF2·GTP ternary complex to form a stable 40 S preinitiation complex (Fig. 8). These findings suggest the
possibility that the 40 S·eIF3·eIF1·eIF1A complex may act as an
intermediate in the binding of the Met-tRNAi·eIF2·GTP
ternary complex to the 40 S subunits to generate the 40 S preinitiation complex. Furthermore, it is possible that both eIF1 and eIF1A, like
eIF3, can bind the 40 S ribosomal subunit prior to message binding.
An important outcome of the present studies is the role of eIF3 and
eIF1A in ribosomal subunit anti-association. Mammalian eIF3 has been
reported to bind free 40 S ribosomal subunits in the absence of
initiator Met-tRNA and other initiation factors (28) and prevent the
Mg2+-dependent association between 40 S and 60 S ribosomal subunits to form 80 S ribosomes (29, 30, 35). We have,
however, observed (20) that, although eIF3 binds stably to free 40 S
ribosomal subunits in the absence of all other initiation components,
the addition of 60 S ribosomal subunits to the 40 S·eIF3 complex
resulted in the release of eIF3 from the 40 S ribosomal subunits with
the concomitant formation of 80 S ribosomes. This was true even when eIF1A was included in the reaction (19, 20). These results suggested
that eIF3 and eIF1A may not be directly involved in the generation of
free ribosomal subunits. The results presented in this report, however,
show that eIF3 does have anti-association factor activity in the
context of the 40 S preinitiation reactions. In the absence of eIF3, 60 S subunits can displace the 40 S subunits present in the 40 S
preinitiation complex and presumably associate with these 40 S subunits
to form 80 S ribosomes (Fig. 3). The presence of eIF3 (along with eIF1A
and eIF1) bound to the 40 S preinitiation complex prevents the
displacement of 40 S subunits from the preinitiation complex by the 60 S subunits (Fig. 3). Thus, under these conditions, 80 S ribosomes
cannot be formed.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3' direction until the 40 S complex encounters the
initiating AUG codon to form the 40 S initiation complex (40 S·eIF3·mRNA·Met-tRNAi·eIF2·GTP). This
reaction requires the participation of three other initiation factors
eIF4F, eIF4A, and eIF4B. Subsequently, the 60 S ribosomal subunit joins
the 40 S complex in a reaction dependent on two other factors, eIF5 and
eIF5B, to form a functional 80 S initiation complex (80 S·mRNA·Met-tRNAi) (for review, see Refs. 1-5).
-globin mRNA, have reported that eIF1 in concert with eIF1A,
promotes stable 40 S complex formation with ribosomes positioned at the
correct AUG codon. The requirements of eIF1 and eIF1A in AUG selection
were also observed by Algire et al. (18) in a reconstituted
yeast translation initiation system containing a 43-nucleotide-long RNA
with an AUG codon in the middle. However, the step at which eIF1 and
eIF1A associate with the 40 S ribosomal subunits remains unclear. Are
these initiation factors recruited following binding of the 40 S
preinitiation complex to the 5'-capped end? Or, are both these factors
recruited to the 40 S ribosomal subunit prior to the binding of the
preinitiation complex to the 5'-end of the mRNA?
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactoside, and grown
for an additional 2 h. The cells were harvested by centrifugation, washed with 0.9% NaCl, quick-frozen in a dry ice/ethanol bath, and
stored at
70 °C.
20 °C. The
yield was about 1 mg of homogeneous protein. eIF1 was monitored at
different purification steps by SDS-PAGE followed by Coomassie Blue
staining as well as by immunoblot analysis using rabbit polyclonal anti-eIF1 antibodies (data not shown).
-D-thiogalactoside, and then
grown for an additional hour. The culture was then supplemented with 5 mCi of [3H]leucine (Amersham Biosciences), and the cells
were allowed to grow with vigorous aeration for an additional hour. The
cells were then harvested by centrifugation, washed with ice-cold 0.9% NaCl, and then quick-frozen. Homogeneous [3H]eIF1 (~800
cpm/pmol of protein) was isolated from the frozen cells using the
purification protocol described above for the isolation of
non-radioactive recombinant eIF1.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]GTP
in lieu of [35S]Met-tRNAi·eIF2·GTP. Under
these conditions, nearly identical molar amounts of 3H and
32P were bound to 40 S ribosomes (data not shown).
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Fig. 1.
Effect of saturating concentrations of eIF1A,
eIF3, and eIF1 on the formation of a stable 40 S preinitiation
complex. The 40 S preinitiation complex was formed and analyzed as
described under "Experimental Procedures." Reaction mixtures
contained Buffer B (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM MgCl2, 2.5 mM 2-mercaptoethanol), 0.6 A260 unit
of 40 S ribosomal subunits, and where indicated, purified rabbit
reticulocyte eIF3 (5 µg), eIF1A (1.0 µg), and eIF1 (0.5 µg).
Following incubation at 37 °C for 4 min, each reaction mixture was
chilled and supplemented with 95 µl of the preformed
[35S]Met-tRNAi·eIF2·GTP ternary complex
containing 3.5 pmol of bound [35S]Met-tRNAi
(66,000 cpm/pmol) (see "Experimental Procedures"). After adjusting
the Mg2+ concentration to 1 mM, they were
incubated a second time at 37 °C for 4 min, chilled, and then
analyzed by sucrose gradient centrifugation in buffers containing 1 mM Mg2+ ("Experimental Procedures").
Initiation factor additions were as follows; , no addition;
,
eIF1;
, eIF3;
, eIF1A;
, eIF1 plus eIF3;
, eIF1A plus eIF3;
, eIF1A plus eIF1;
, eIF1A plus eIF1 plus eIF3. The
ascending arrows indicate 35S radioactivity
recovered at the top of each gradient tube corresponding to unreacted
free [35S]Met-tRNAi and unreacted
[35S]Met-tRNAi·eIF2·GTP ternary complex.
The position of the 40 S particle was determined in a parallel
gradient.
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Fig. 2.
Effect of limiting concentrations of eIF1 and
eIF1A on the production of the stable 40 S preinitiation complex.
The 40 S preinitiation complex was formed as described under the legend
to Fig. 1 and "Experimental Procedures," except that each reaction
contained the indicated levels of either eIF1 ( ), eIF1A (
), or
eIF1 plus eIF1A (
). eIF3 was not added to these reactions. Each
reaction mixture was analyzed by sucrose gradient centrifugation
("Experimental Procedures"). Ordinate: at each indicated
concentration of the initiation factors, the total amount (picomoles)
of 40 S preinitiation complex formed was calculated.
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Fig. 3.
Effect of 60 S ribosomal subunits on the
stability of the 40 S preinitiation complex. Two sets of reaction
mixtures were used to form the 40 S preinitiation complex as described
under legend to Fig. 1 and "Experimental Procedures," except that
0.3 A260 unit of 40 S ribosomal subunits was
used. Each reaction contained 3.5 pmol of preformed
[35S]Met-tRNAi·eIF2·GTP ternary complex
(60,000 cpm/pmol) and, where indicated, eIF1A (200 ng), eIF1 (150 ng),
and eIF3 (3 µg). Following incubation to form the 40 S preinitiation
complex, only one set of reaction mixtures was supplemented with 1.2 A260 units of 60 S ribosomal subunits. The
Mg2+ concentration of all reaction mixtures was then raised
to 5 mM. After incubation at 37 °C for an additional 4 min, the mixtures were chilled on ice, and the amount of stable 40 S
preinitiation complex remaining in each reaction mixture was analyzed
by sucrose gradient centrifugation ("Experimental Procedures").
Ordinate: the total amount of
[35S]Met-tRNAi bound to the 40 S region in
each reaction. It should be noted that the results presented in this
figure are an average of three independent experiments.
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Fig. 4.
Association of eIF1A and eIF1 with the 40 S
preinitiation complex. A, two reaction mixtures were
prepared to measure formation of the 40 S preinitiation complex. Each
contained 3 µg of eIF3, 200 ng of eIF1A, and 150 ng of eIF1. In one
reaction mixture, 3.5 pmol of
[35S]Met-tRNAi·eIF2·GTP ternary complex
(50,000 cpm/pmol) was added to determine the amount of the 40 S
preinitiation complex formed. In the other reaction,
[3H]eIF1 (820 cpm/pmol) replaced unlabeled eIF1, and the
specific activity of [35S]Met-tRNAi in the
ternary complex added was only 50 cpm/pmol. The specific activity of
the [35S]Met-tRNAi in this reaction was
intentionally kept below the level of detection in the analysis.
Following incubation to form the 40 S preinitiation complex, each
reaction mixture was sedimented through sucrose gradients
("Experimental Procedures"). Fractions were collected from the
bottom of each tube, and the amount of 35S or
3H radioactivity was determined by liquid scintillation
counting. Control reaction mixtures (not shown) in which either 40 S
subunits or the ternary complex was omitted were also analyzed. Under
these conditions, no 3H radioactivity was detected in the
40 S region. B, the 40 S preinitiation complex was formed in
the presence of 3 µg of eIF3, 200 ng of eIF1A, and 150 of ng eIF1
("Experimental Procedures") except that
[3H]Met-tRNAi (10,000 cpm/pmol) and
[35S]eIF1A (1800 cpm/pmol) replaced
[35S]Met-tRNAi and unlabeled eIF1A. Following
incubation to form the 40 S preinitiation complex, the chilled reaction
mixtures were analyzed by sucrose gradient centrifugation. Fractions
(250 µl) were collected from the bottom of each tube, and the amount
of 35S and 3H radioactivity in each fraction
was determined by liquid scintillation counting. Control reaction
mixtures (not shown) in which either 40 S subunits or
[3H]Met-tRNAi was omitted were also analyzed.
In these reaction mixtures, no 3H or 35S
radioactivity was detected in the 40 S region. C, aliquots
(30 µl) of gradient fractions from reactions described in
B were subjected to Western blot analysis using chicken
anti-rabbit eIF3 antibodies as a probe.
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Fig. 5.
Requirements for the binding of eIF1 and
eIF1A to the 40 S preinitiation complex. A, synthesis
of the 40 S preinitiation complex was carried out as described under
"Experimental Procedures" except that 150 ng of
[3H]eIF1 (820 cpm/pmol) and
[35S]Met-tRNAi (50 cpm/pmol) were added in
lieu of unlabeled eIF1 and [35S]Met-tRNAi
(60,000 cpm/pmol), respectively. As in Fig. 4, the specific
activity of [35S]Met-tRNAi was kept too low
to be detected in this analysis. In addition, as indicated, 200 ng of
eIF1A and 3 µg of eIF3 were present in each reaction mixture.
Following incubation at 37 °C for 4 min to form the 40 S
preinitiation complex, products were analyzed by sucrose gradient
centrifugation. The amount of [3H]eIF1 sedimenting at the
40 S region was determined. For each reaction mixture the amount of 40 S preinitiation complex formed was monitored by forming a 40 S
preinitiation complex in which unlabeled eIF1 and
[35S]Met-tRNAi (60,000 cpm/pmol) were used
and analyzed in a parallel gradient. B, reaction mixtures
were prepared as described under "Experimental Procedures," except
that 200 ng of [35S]eIF1A (1800 cpm/pmol) and
[3H]Met-tRNAi (10,000 cpm/pmol) replaced
unlabeled eIF1A and [35S]Met-tRNAi (60,000 cpm/pmol), respectively. In addition, each reaction mixture contained,
where indicated, 150 ng of eIF1 and 3 µg of eIF3. Following formation
of the 40 S preinitiation complex and analysis by sucrose gradient
centrifugation, the amount of [3H]Met-tRNAi
and [35S]eIF1A bound to the 40 S region was determined.
It should be noted that the results presented in this figure are an
average of three independent experiments.
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Fig. 6.
Binding of eIF1 and eIF1A to the 40 S
preinitiation complex: analysis by Sephadex G-75 gel filtration.
Two sets of reaction mixtures (each containing three reactions) were
prepared for the formation of the 40 S preinitiation complex. The
complete system in each set was prepared as described under the legend
to Fig. 5, except that in one set [3H]eIF1 (see Fig.
5A) was used, whereas the other used
[35S]eIF1A (see Fig. 5B). Various omissions
were as indicated. Following 40 S preinitiation complex formation
("Experimental Procedures"), each reaction mixture was passed
through a 12-ml bed volume column of Sephadex G-75 ("Experimental
Procedures"). The eluted fractions were assayed for 3H or
35S radioactivity. The positions at which the 40 S
preinitiation complex, free [3H]eIF1, and free
[35S]eIF1A eluted from the column were determined
separately in the same column and are shown.
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Fig. 7.
Binding of eIF1 and eIF1A to free 40 S
ribosomal subunits. Two sets of reaction mixtures (each 150 µl)
containing buffer B (see legend to Fig. 1) and 1.8 A260 units of 40 S subunits were prepared. In
one set of reaction mixtures, the complete system contained 20 µg of
eIF3, 560 ng of [3H]eIF1, and 600 ng of unlabeled eIF1A,
whereas in the other set, the complete system contained 20 µg of
eIF3, 625 ng of [35S]eIF1A, and 620 ng of unlabeled eIF1.
In each set the omissions of one or more reaction components were as
indicated. Following incubation at 37 °C for 4 min, the reactions
were chilled on ice and analyzed by Sephadex G-75 gel filtration.
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Fig. 8.
Formation of the 40 S preinitiation complex
using isolated 40 S·eIF3·eIF1·eIF1A complex. Two reaction
mixtures (each 150 µl) containing buffer B, 1.8 A260 units of 40 S ribosomal subunits, and 20 µg of eIF3 were prepared. To one reaction mixture, 560 ng of
[3H]eIF1 and 600 ng of unlabeled eIF1A were added,
whereas the other received 625 ng of [35S]eIF1A and 620 ng of unlabeled eIF1. Following incubation at 37 °C for 4 min, the
products were subjected separately to Sephadex G-75 gel filtration. 40 S ribosome-bound [3H]eIF1 or 40 S ribosome-bound
[35S]eIF1A eluting in the excluded fractions were pooled
separately. The pooled fraction containing [3H]eIF1 was
treated with [35S]Met-tRNAi·eIF2·GTP
ternary complex (50 cpm/pmol), whereas that containing
[35S]eIF1A was treated with
[3H]Met-tRNAi·eIF2·GTP ternary complex
(10,000 cpm/pmol). Following incubation at 37 °C for 4 min to form
the 40 S preinitiation complex, the products were analyzed by sucrose
gradient centrifugation. Aliquots of each gradient were assayed for
3H and 35S radioactivity to determine the level
of 40 S preinitiation complex formed as well as [3H]eIF1
or [35S]eIF1A bound to the 40 S preinitiation complex.
Aliquots of each fraction were subjected to Western blot analysis using
chicken anti-eIF3b (p116) antibodies (bottom panel).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Jerard Hurwitz of Memorial Sloan-Kettering Cancer Center, New York and Dr. Dennis Shields of the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by Grant GM15399 from the National Institutes of Health (NIH) and by Cancer Core Support Grant P30CA13330 from the NCI, NIH.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: Dept. of Genetics, Harvard Medical School,
Boston, MA 02125.
§ To whom correspondence should be addressed. Tel.: 718-430-3505; Fax: 718-430-8567; E-mail: maitra@aecom.yu.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M210357200
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ABBREVIATIONS |
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The abbreviation used is: eIF, eukaryotic (translation) initiation factor.
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REFERENCES |
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