(Received for publication, October 2, 1996, and in revised form, December 9, 1996)
From the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461
We have used an efficient in vitro translation initiation system to show that the mammalian 17-kDa eukaryotic initiation factor, eIF1A (formerly designated eIF-4C), is essential for transfer of the initiator Met-tRNAf (as Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA to form the 40 S preinitiation complex (40 S·Met-tRNAf·eIF2·GTP). Furthermore, eIF1A acted catalytically in this reaction to mediate highly efficient transfer of the Met-tRNAf·eIF2·GTP ternary complex to 40 S ribosomal subunits. The 40 S complex formed was free of eIF1A indicating that its role in 40 S preinitiation complex formation is not to stabilize the binding of Met-tRNAf to 40 S ribosomes. Additionally, the eIF1A-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. In contrast to other reports, we found that eIF1A plays no role either in the subunit joining reaction or in the generation of ribosomal subunits from 80 S ribosomes. Our results indicate that the major function of eIF1A is to mediate the transfer of Met-tRNAf to 40 S ribosomal subunits to form the 40 S preinitiation complex.
The initiation of translation in eukaryotic cells occurs by a
sequence of well defined partial reactions that require a large number
of specific proteins called eukaryotic translation initiation factors
(eIFs)1 (for reviews, see Refs. 1-5).
According to the current accepted view of initiation (4), the first
step involves the binding of GTP and the initiator
Met-tRNAf to initiation factor eIF2 to form the
Met-tRNAf·eIF2·GTP ternary complex, which is then
transferred to the 40 S ribosomal subunit in a reaction that is
stimulated approximately 2-fold by the multi-subunit initiation factor
eIF3 to form the 40 S preinitiation complex
(eIF3·40 S·Met-tRNAf·eIF2·GTP). The 40 S
preinitiation complex then binds to the capped 5 end of mRNA in a
reaction requiring eIF4F, eIF4A, and eIF4B and begins scanning the
mRNA until it selects the appropriate initiation AUG codon where
the 40 S complex is positioned on the mRNA through codon-anticodon
base pairing to form the 40 S initiation complex (40 S·mRNA·eIF3·Met-tRNAf·eIF2·GTP).
Initiation factor eIF5 then interacts with the 40 S initiation complex
resulting in the hydrolysis of 40 S subunit-bound GTP. The hydrolysis
leads to the release of the initiation factors bound to the complex and
the concomitant joining of the 60 S ribosomal subunit to the 40 S
complex to form the functional 80 S initiation complex (1-5). It has
been reported that a 17-kDa protein, eIF1A (formerly called eIF-4C),
stimulates formation of both the 40 S and 80 S initiation complexes
about 2- and 3-fold, respectively (6-9). The Saccharomyces
cerevisiae homologues of eIF1A and another low molecular weight
initiation factor, eIF1, have been shown to be essential genes (10-12)
that play important roles in translation initiation. However, the
function of these proteins in the initiation pathway is not clearly
understood.
In our laboratory, we have been studying each partial reaction of the initiation pathway separately to define the requirements of each reaction. We have previously shown (13) that when the Met-tRNAf·eIF2·GTP ternary complex was incubated with 40 S ribosomal subunits and AUG at an elevated Mg2+ concentration of 5 mM (the physiological Mg2+ concentration is about 1-2 mM), the ternary complex was efficiently transferred to 40 S ribosomal subunits to form the stable 40 S initiation complex (40 S·AUG·Met-tRNAf·eIF2·GTP) without the requirement for any additional initiation factors. This 40 S initiation complex served as an efficient substrate for the eIF5-mediated joining of 60 S ribosomal subunits to form a functional 80 S initiation complex (13-16). These reactions have been utilized in our laboratory to purify and characterize eIF5 from both mammalian cells (14-18) and S. cerevisiae (19, 20). However, we have now observed that when Met-tRNAf·eIF2·GTP ternary complex was incubated with 40 S ribosomal subunits and AUG at a more physiological Mg2+ concentration of 1-2 mM, little or no transfer of Met-tRNAf to 40 S subunits occurred. During the course of purification of initiation factors from 0.5 M KCl ribosomal wash proteins from rabbit reticulocyte lysates, we observed that the addition of a protein fraction eluting from the phosphocellulose column with 1 M KCl to the 40 S initiation reaction mixture at 1 or 2 mM Mg2+ supported efficient transfer of the ternary complex to 40 S ribosomal subunits. Purification and characterization of the stimulatory factor present in the 1 M phosphocellulose eluate showed that the activity responsible for this transfer is the 17-kDa-eIF1A. This result was surprising in view of the previous reports (6-8) that eIF1A stimulated 40 S initiation complex formation only 1.5-2-fold.
In this paper, we have used homogeneous eIF1A isolated from rabbit reticulocyte lysates as well as bacterially expressed recombinant eIF1A to investigate the role of this protein in the formation of 40 S and 80 S ribosomal initiation complexes. The function of this protein as a ribosomal subunit anti-association factor has also been studied. The results we have obtained demonstrate that eIF1A is essential for the transfer of Met-tRNAf (as Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomal subunits prior to the binding of mRNA to form the 40 S preinitiation complex. In contrast to reports from other laboratories (6-9), we find that eIF1A has no demonstrable role either in the joining of 60 S ribosomal subunits to the 40 S initiation complex or in the dissociation of 80 S ribosomes.
The preparation of
35S- or 3H-labeled rabbit liver
Met-tRNAf (10,000-30,000 cpm/pmol) and ribosomal subunits
from Artemia salina eggs were as described previously (21,
22). Homogeneous recombinant rat eIF5 was isolated from
Escherichia coli as described (23). Initiation factors eIF2
and eIF3 were purified from 300 ml of rabbit reticulocyte lysates
(Green Hectares Co.) as follows. The preparation of crude ribosomal 0.5 M KCl wash proteins and their subsequent fractionation by
successive stepwise elution from DEAE-cellulose and phosphocellulose
columns followed by FPLC-Mono Q (HR5/5) chromatography were as
described previously for the isolation of rabbit reticulocyte eIF5 from
this laboratory (16). During the Mono Q gradient step, eIF2 eluted at
~270 mM KCl while eIF3 eluted at ~400 mM
KCl. Fractions containing eIF2 activity were pooled and further
purified by FPLC-Mono S chromatography by an adaptation of the
procedure of Dholakia and Wahba (24). Purified eIF2 exhibited three
major polypeptide bands, corresponding to the ,
, and
subunits of eIF2 (25). For further purification of eIF3, the pooled
Mono Q-eIF3 fraction was concentrated to about 1 ml by Centricon-30
filtration and then purified by centrifugation at 40,000 rpm in two
15-40% (w/v) glycerol gradients containing 20 mM Tris-Cl,
pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, and
650 mM KCl for 20 h in an SW41 rotor at 2 °C. Fractions containing eIF3 activity were pooled, dialyzed against buffer
A (20 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol,
0.1 mM EDTA, and 10% glycerol) containing 100 mM KCl to reduce the KCl concentration to 100 mM, and then fractionated on a FPLC-Mono S column (1-ml bed
volume). eIF3 activity eluted at about 400 mM KCl. Active fractions were pooled, dialyzed for about 5 h against buffer A containing 55% glycerol and 100 mM KCl, and then stored in
small aliquots at
70 °C.
Crude 0.5 M
KCl ribosomal wash proteins (120 mg) prepared from 300 ml of rabbit
reticulocyte lysates (16) was loaded onto a 40-ml bed volume of a
DEAE-cellulose column that had been equilibrated in buffer A + 100 mM KCl. After washing the column with this buffer, the
bound proteins were eluted with buffer A + 300 mM KCl and dialyzed against buffer A + 75 mM KCl to reduce the ionic
strength of the fraction to that of buffer A + 100 mM KCl.
The dialyzed DEAE-cellulose eluate was then applied to a 12-ml bed
volume of a phosphocellulose column, equilibrated in buffer A + 100 mM KCl. After washing the column with this buffer, the
bound proteins were eluted successively with 40-ml volumes of buffer A
containing increasing concentrations of KCl as follows: (a)
300 mM KCl, (b) 650 mM KCl, and
(c) 1 M KCl. The phosphocellulose, 1 M KCl eluate (0.5 mg of protein) was dialyzed against
buffer A + 100 mM KCl to reduce the ionic strength to that
of buffer A + 100 mM KCl and then applied to 1-ml bed
volume FPLC-Mono Q column. The column was washed with 5 ml of this
buffer, and bound proteins were then eluted with two consecutive linear
gradients in buffer A (1 ml/min) as follows: 100 mM 250 mM KCl (total volume 5 ml), followed by 250 mM
600 mM KCl (total volume 25 ml). Fractions containing eIF1A activity (eluting at about 340 mM KCl) were pooled
and stored in small aliquots at
70 °C.
The open reading frame
of eIF1A cDNA (10) was synthesized by reverse
transcription-polymerase chain reaction of HeLa poly(A)+
RNA using Life Technologies, Inc. kit. The primer sequences used were
as follows: N terminus, 5-dC
CCCAAGAATAA-3
; and C terminus, 5
-dGC
TTAGATGTCATCAATATCTTC-3
.
The 460-nucleotide long polymerase chain reaction product was sequenced to ensure error-free DNA synthesis and cloned into the
NdeI/EcoRI sites of pET-5a plasmid (26)
(Novagen). This pET-5a-eIF1A expression vector in which the eIF1A
coding sequence is under the transcriptional control of a T7 RNA
polymerase promoter was used to transform E. coli BL21 (DE3)
cells (Novagen) that contain T7 RNA polymerase gene in its chromosome
under the control of lacUV5 promoter (26). A single colony
isolated as an ampicillin-resistant transformant was grown to
mid-logarithmic phase in 5 ml of LB medium (27) containing 50 µg/ml
ampicillin. The cells were then diluted 1:1000 into 1 liter of LB
medium containing 50 µg/ml ampicillin and grown at 37 °C to
A600 of about 1.2, induced with 1 mM
isopropyl-
-D-thiogalactoside, and grown for an
additional 3 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.
For purification of recombinant eIF1A, frozen E. coli cells
(5 g) were disrupted by sonication, and the post-ribosomal supernatant was prepared as described previously for the isolation of recombinant eIF5 from overproducing E. coli cells (23). The
post-ribosomal supernatant (75 mg of protein in 10-ml total volume) was
adjusted to 100 mM KCl by the addition of 2 M
KCl and then loaded onto a 20-ml bed volume of DEAE-cellulose column
equilibrated in buffer A + 100 mM KCl. After washing the
column with the same buffer to remove unabsorbed proteins, bound
proteins were eluted with buffer A + 300 mM KCl. The eluted
proteins (24 mg) were directly applied to a 5-ml phosphocellulose
column equilibrated in buffer A + 300 mM KCl. The column
was then washed with buffer A + 450 mM KCl until
A280 was below 0.1. Bound eIF1A was then eluted
from the column with buffer A + 1 M KCl. The 1 M KCl eluate was dialyzed against buffer A + 100 mM KCl and then subjected to FPLC-Mono Q (1-ml column)
chromatography similar to that described above for rabbit
reticulocyte-eIF1A. Fractions containing eIF1A (eluting at about 350 mM KCl) were pooled and stored in small aliquots at
70 °C. The yield was about 3 mg of homogeneous protein. eIF1A was
monitored at different purification steps by SDS-polyacrylamide gel
electrophoresis followed by Coomassie Blue staining as well as by
immunoblot analysis using rabbit polyclonal anti-eIF1A antibodies as
the probe (data not shown).
E.
coli BL21 (DE3) harboring the pET-5a-eIF1A expression plasmid was
grown at 37 °C in 500 ml of M9 medium (27) containing 1 mM MgSO4 and 50 µg/ml ampicillin to
A600 of about 1.0. The cells were then pelleted
by centrifugation and resuspended in 500 ml of prewarmed M9 medium
containing 0.4 mM MgSO4 and 50 µg/ml
ampicillin. Cells were grown for 1 h at 37 °C with vigorous
aeration, induced with 1 mM
isopropyl--D-thiogalactoside, and then grown for an additional 150 min. The culture was then supplemented with 5 mCi [35S]methionine and [35S]cysteine
(Tran35S-label, DuPont NEN), and the cells allowed to grow
with vigorous aeration for an additional 30 min. Cells were then
harvested by centrifugation, washed with ice-cold 50 mM
Tris-HCl, pH 8.0, followed by an additional washing with 0.9% NaCl,
and then quick-frozen. Homogeneous 35S-eIF1A (~3,000
cpm/pmol protein) was isolated from the frozen cells using the
purification protocol described above for the isolation of
unlabeled recombinant eIF1A.
Approximately 60 µg of purified rabbit reticulocyte eIF1A were subjected to SDS-polyacrylamide (10%) gel electrophoresis. The 17-kDa-eIF1A polypeptide was excised with a scalpel and used as an antigen for the preparation of antisera against eIF1A in mature rabbits, following a protocol similar to that used previously for anti-eIF5 antibodies (15). The antibodies were affinity purified against purified rabbit reticulocyte stimulatory protein (eIF1A) by an adaptation of the procedure of Olmsted (28) as described for affinity purification of anti-eIF5 antibodies by Ghosh et al. (15). Immunoblot analysis of eIF1A was performed using rabbit polyclonal anti-eIF1A antibodies as probes following a procedure previously used in this laboratory for immunoblot analysis of eIF5 (15).
Assay of eIF1A ActivityeIF1A activity was routinely assayed for its ability to mediate the transfer of 35S- or 3H-labeled Met-tRNAf from Met-tRNAf·eIF2·GTP ternary complex to 40 S ribosomal subunits in the presence of 1 mM Mg2+. Reactions were carried out in two stages as follows. In stage 1, 0.8-1.2 µg of purified eIF2, 8 pmol of [35S]Met-tRNAf, and 6 µM GTP were incubated in reaction mixtures (50 µl) containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM 2-mercaptoethanol, and 4 µg of nuclease-free bovine serum albumin for 4 min at 37 °C to promote formation of the [35S]Met-tRNAf·eIF2·GTP ternary complex. In stage 2, the above reaction mixtures were supplemented with eIF1A (10-200 ng of protein), 0.05 A260 unit of AUG codon, 0.6 A260 unit of 40 S ribosomal subunits, and MgCl2 (1 mM final concentration). Following incubation at 37 °C for 4 min, reaction mixtures (125 µl each) were chilled in ice water and layered onto 7.5-30% (w/v) sucrose gradients (5 ml each) containing 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 100 mM KCl, 5 mM 2-mercaptoethanol and centrifuged at 48,000 rpm for 105 min in a SW 50.1 rotor. Fractions (200-350 µl) were collected from the bottom of each tube, and the radioactivity was measured by counting in Aquasol in a liquid scintillation spectrometer. Under the conditions of this assay, formation of the 40 S initiation complex was directly proportional to the amount of eIF1A added. The efficiency of 40 S initiation complex formation was calculated relative to [35S]Met-tRNAf·eIF2·GTP ternary complex formed in stage 1 incubation.
Isolation of the 40 S Initiation ComplexAn initiation
reaction mixture (175 µl) was prepared and incubated as described
above under "Assay of eIF1A Activity" with the following
modifications. During the formation of the ternary complex (stage 1 incubation), 40 pmol of [35S]Met-tRNAf
(20,000 cpm/pmol), 12 µg of purified eIF2, and 18 µM
GTP were added, and during the formation of the 40 S initiation complex (stage 2), 1.8 A260 units of 40 S
ribosomal subunits, 0.12 A260 unit of AUG, and
0.6 µg of purified recombinant eIF1A were added. After incubation to
form the 40 S initiation complex, the chilled reaction mixture was
subjected to 7.5-30% sucrose gradients as described above. Fractions
containing 40 S initiation complex, free of unreacted reaction
components, were pooled, divided into small aliquots, and stored at
70 °C until use.
eIF3 activity was measured by its ability to bind to 40 S ribosomal subunits and stimulate between 2 and 4-fold the AUG-dependent binding of [35S]Met-tRNA·eIF2·GTP ternary complex to 40 S ribosomal subunits in the presence of 1 mM Mg2+. This assay is similar to that described above for eIF1A except that eIF3 was added in lieu of eIF1A. Protein was determined by the method of Bradford (29) using reagents obtained from Bio-Rad.
Formation of the 40 S initiation complex can be measured using either natural mRNA (e.g. globin mRNA) or the trinucleotide codon, AUG, as a template. In reactions containing globin mRNA, formation of the 40 S initiation complex (40 S·mRNA·Met-tRNAf) involves two separate reactions that occur in the following sequence. Initially, Met-tRNAf·eIF2·GTP ternary complex binds to 40 S ribosomal subunits (in the absence of mRNA) to form the 40 S preinitiation complex. This is then followed by the scanning of the mRNA by the 40 S preinitiation complex to locate and then recognize the initiation AUG codon to form the 40 S initiation complex. In contrast, the AUG-directed system directly measures the initial transfer of Met-tRNAf to the 40 S ribosomes leading to the formation of the 40 S preinitiation complex. In this reaction, AUG acts solely to stabilize the binding of Met-tRNAf to the 40 S ribosomal subunit by codon-anticodon base pairing to form the stable 40 S initiation complex (40 S·AUG·Met-tRNAf·eIF2·GTP). In this system, the requirements for the mRNA-binding proteins eIF4A, eIF4B, and eIF4F, which are essential for the scanning of mRNA, are bypassed.
The requirements for the initial transfer of
Met-tRNAf·eIF2·GTP ternary complex to 40 S ribosomes
prior to mRNA binding were examined. For this purpose, preformed
[35S]Met-tRNAf·eIF2·GTP ternary complex
was incubated with 40 S ribosomal subunits in the presence of AUG
codon, and the reaction products were then analyzed by sucrose gradient
centrifugation. When the initiation reaction was carried out at an
elevated Mg2+ concentration of 5 mM,
[35S]Met-tRNAf (presumably as
Met-tRNAf·eIF2·GTP·ternary complex) was nearly
quantitatively transferred to 40 S ribosomes without the requirement
of any additional protein factors (Fig. 1 and Refs.
13-16). In contrast, when the initiation reaction was carried out at
the more physiological Mg2+ concentration of 1 or 2 mM (only the results with 1 mM Mg2+
are shown), little or no 40 S initiation complex was formed (Fig. 1).
To identify one or more initiation factor(s) that may be required for
the formation of the 40 S preinitiation complex at 1-2 mM Mg2+, we fractionated the 0.5 M KCl wash
proteins derived from rabbit reticulocyte polysomes through successive
DEAE and phosphocellulose chromatographic steps as described under
"Experimental Procedures." As shown in Fig. 1, supplementation of
the components of the 40 S initiation reaction with the protein
fraction eluting from phosphocellulose column with 1 M KCl
resulted in highly efficient transfer of
[35S]Met-tRNAf to 40 S ribosomal subunits.
While only 0.15 pmol of Met-tRNAf was bound to 40 S
ribosomes in the absence of the phosphocellulose fraction, the addition
of this fraction resulted in the binding of 2.4 pmol of
Met-tRNAf to 40 S ribosomes (a stimulation of nearly 18-fold). Initiation factor eIF3, in agreement with reports from other
laboratories (7-9), also stimulated this transfer reaction approximately 3-fold. However, the efficiency of transfer of
Met-tRNAf to 40 S ribosomes mediated by the stimulatory
protein was far greater than that obtained with an excess of eIF3 (Fig.
1). The stimulatory activity present in the phosphocellulose fraction was further purified by gradient elution from an FPLC-Mono Q column (see "Experimental Procedures"). Analysis of this activity by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining showed a single polypeptide band of about 17 kDa (Fig. 2, panel A).
The apparent molecular weight of the stimulatory protein and its strong
affinity for phosphocellulose are properties similar to those of the
initiation factor eIF1A (formerly eIF-4C) (4). Mass spectrometric
analysis (not shown) showed that the purified fraction containing the
stimulatory activity contained one major protein species of
Mr = 16,332. This value is identical to the molecular weight of eIF1A lacking the N-terminal methionine as deduced
from the human eIF1A-cDNA clone (10). Further confirmation that the
stimulatory activity purified here was indeed eIF1A was derived from
the following observations. First, recombinant eIF1A was purified to
apparent electrophoretic homogeneity from overproducing E. coli cells as described under "Experimental Procedures." The homogeneous recombinant eIF1A protein (Fig. 2, panel A, lane
b) was as active as the stimulatory protein purified from rabbit reticulocytes lysates in mediating the transfer of
Met-tRNAf to 40 S ribosomal subunits (Fig.
3, panels A and B). eIF3 was far less active
than eIF1A in this transfer reaction. Even in the presence of excess
eIF3, the amount of Met-tRNAf transferred never reached the
value obtained with limiting concentrations of eIF1A (Fig. 3,
panel B). Second, specific antisera were raised in rabbits against purified denatured rabbit reticulocyte stimulatory protein (Mono Q fraction) and affinity purified against purified stimulatory protein. The affinity purified antibodies reacted strongly with recombinant eIF1A (Fig. 2, panel B, lane d) further
confirming that the stimulatory activity present in the reticulocyte
Mono Q fraction was indeed eIF1A. Taken together, these results
strongly suggest that eIF1A is essential for the transfer of
Met-tRNAf to 40 S ribosomal subunits at the physiological
Mg2+ concentration of 1 mM.
Further Characterization of eIF1A-mediated Transfer of Met-tRNAf to 40 S Ribosomal Subunits
We
characterized the eIF1A-mediated binding of Met-tRNAf to
40 S ribosomes in greater detail. In addition to eIF1A, the binding reaction was completely dependent on eIF2 (Fig. 3, panel A)
and was markedly decreased by omitting GTP from the reaction (data not
shown; only 0.12 pmol of the 40 S initiation complex was formed in the
absence of GTP). Mammalian eIF1A, like the homologous protein from
wheat germ (30), was found to be heat stable. Incubation of recombinant
eIF1A at 90 °C for 4 min had no effect on its ability to mediate the
transfer of Met-tRNAf to 40 S ribosomes (data not shown).
Furthermore, in agreement with the requirements of eIF2 and GTP for the
eIF1A-mediated transfer of Met-tRNAf to 40 S ribosomes, we
observed that when the 40 S incubation mixture contained
[3H]Met-tRNAf and [-32P]GTP,
eIF1A mediated the transfer of nearly identical molar amounts of both
3H and 32P to 40 S particles (Fig.
4A, upper panel). Immunoblot analysis of
gradient fractions for eIF2 using rabbit anti-eIF2 antibodies showed
that eIF2 also co-sedimented with the 40 S particles (Fig. 4A,
lower panel). In the absence of eIF1A, insignificant amounts of
[3H]Met-tRNAf and [32P]GTP were
transferred to 40 S ribosomes, and no immunoreactive eIF2 polypeptides
sedimented with the 40 S ribosomes (Fig. 4B, upper and
lower panels). These results indicate that eIF1A mediated the transfer of the entire Met-tRNAf·eIF2·GTP ternary
complex to 40 S ribosomes. In other experiments (data not presented)
when a preformed [3H]Met-tRNA·eIF2·GTP ternary
complex was first isolated by Sephadex G-75 gel filtration and then
incubated at 1 mM Mg2+ with 40 S ribosomes,
AUG codon, and eIF1A, the entire ternary complex was transferred to
40 S ribosomal subunits to form the 40 S initiation complex.
eIF1A-mediated Transfer of Met-tRNAf to 40 S Ribosomes in the Absence of AUG Codon
Experiments presented thus far
measured the ability of eIF1A to mediate the transfer of the
Met-tRNAf·eIF2·GTP ternary complex to 40 S ribosomes
in the presence of AUG. To demonstrate that eIF1A is indeed required
for the initial transfer reaction and AUG plays a passive role in this
reaction presumably by stabilizing the binding of the transferred
Met-tRNAf to 40 S ribosome, the preformed
[35S]Met-tRNAf·eIF2·GTP ternary complex
was incubated with 40 S ribosomes and eIF1A in the absence of the AUG
codon in reactions containing 1 mM MgCl2. The
products formed were then analyzed by sedimentation through sucrose
gradients in buffers containing 1 or 5 mM
MgCl2. Elevated Mg2+ concentration is known to
stabilize ribosomal binding of aminoacyl-tRNAs and thus has been used
to analyze 40 S and 80 S initiation complexes by sucrose gradient
centrifugation (7, 8). As shown in Fig. 5, low levels of
Met-tRNAf bound to 40 S ribosomes were detected in
gradients containing 1 mM Mg2+, in the presence
or absence of eIF1A. In contrast, when analysis was carried out in
sucrose gradients containing 5 mM Mg2+,
[35S]Met-tRNAf bound to 40 S ribosomes was
readily observed (Fig. 5). The binding of Met-tRNAf to
40 S ribosomes under these conditions was still dependent on the
presence of both eIF1A and eIF2 (Fig. 5) as well as on GTP in the
reaction (data not shown). These results demonstrate that eIF1A
mediates the transfer of Met-tRNAf (as Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomes
prior to the binding of mRNA to form the 40 S preinitiation
complex.
Fate of eIF1A during Formation of the 40 S Preinitiation and Initiation Complexes
To determine the fate of eIF1A during
factor-dependent formation of the 40 S initiation complex,
the preformed [35S]Met-tRNA·eIF2·GTP ternary complex
was incubated with 40 S ribosomal subunits, AUG codon, and eIF1A at 1 mM Mg2+, and the reaction mixture was subjected
to sucrose gradient centrifugation. Under these conditions,
[35S]Met-tRNAf, as expected, sedimented at a
position characteristic of 40 S ribosome-bound Met-tRNAf
(Fig. 6A, upper panel). Western blot analysis
of the gradient fractions with anti-eIF1A antibodies, however, did not
detect eIF1A in the region of the gradient where 40 S ribosome-bound
[35S]Met-tRNAf sedimented. eIF1A sedimented
near the top of the gradient at the position expected for a protein
with an Mr value of about 17,000 (Fig. 6A,
lower panel).
Experiments were also carried out in which a 40 S preinitiation complex was formed by incubating the [3H]Met-tRNAf·eIF2·GTP ternary complex with 40 S ribosomes and 35S-eIF1A (prepared as described under "Experimental Procedures") at 1 mM Mg2+ in the absence of AUG. The reaction products were then subjected to Sephadex G-75 gel filtration (Fig. 6B). [3H]Met-tRNAf bound to 40 S ribosomes eluted from the column in the excluded material. However, insignificant (<0.01 pmol) amounts of 35S (due to 35S-eIF1A) were detected in the excluded fraction (Fig. 6B). Nearly all of the 35S radioactivity eluted from the column at the position expected for free eIF1A protein of Mr = 17,000 (Fig. 6B). These results indicate that although eIF1A is essential for the transfer of Met-tRNAf (as Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomes, eIF1A is not included in the resultant 40 S preinitiation complex and thus does not contribute to the stability of the 40 S preinitiation complex once it is formed.
Catalytic Reutilization of eIF1A in the Formation of the 40 S Preinitiation ComplexOur observation that while eIF1A is
essential for the transfer of Met-tRNAf to 40 S ribosomes
but is not associated with the resultant 40 S preinitiation complex
suggested that the factor might be acting catalytically in the
initiation reaction. To investigate this possibility, we measured
eIF1A-mediated transfer of Met-tRNAf to 40 S ribosomes in
the presence or absence of the AUG codon using a higher molar ratio of
the substrate Met-tRNAf·eIF2·GTP ternary complex to
eIF1A than that used in the experiments presented thus far. The
kinetics of transfer of Met-tRNAf to 40 S ribosomes at 37 and at 16 °C are shown in Fig. 7. At 37 °C, the
transfer reaction was complete in 2 min. In contrast, at 16 °C, the
reaction proceeded more slowly reaching a plateau at about 10 min. This was true regardless of the presence or absence of AUG in the reaction (Fig. 7). More importantly, under all the conditions of the in vitro assay described in Fig. 7, 60 ng of eIF1A (equivalent to about 3.5 pmol of protein) mediated the transfer of nearly 13-15 pmol
of Met-tRNAf to 40 S ribosomes. These results clearly show catalytic reutilization of eIF1A in the formation of the 40 S preinitiation complex.
Effect of eIF1A on eIF5-mediated Joining of 60 S Ribosomal Subunits to the 40 S Initiation Complex
Our ability to isolate
the 40 S initiation complex free of eIF1A allowed us to address the
questions (i) whether the 40 S initiation complex formed under these
conditions is competent to join 60 S ribosomal subunits in the
presence of eIF5 to form a functional 80 S initiation complex, and
(ii) the effect of eIF1A on this subunit joining reaction. For this
purpose, the 40 S initiation complex containing bound
[35S]Met-tRNAf was formed in the presence of
eIF1A, and the complex was isolated free of unreacted reaction
components by sucrose density gradient centrifugation. Western blotting
confirmed that the 40 S initiation complex was free of eIF1A (data not
shown here, see Fig. 6). Incubation of the isolated 40 S initiation complex with 60 S ribosomal subunits and eIF5 resulted in the quantitative formation of the 80 S initiation complex (Fig.
8, panel A). Formation of the 80 S
initiation complex was completely dependent on the presence of both
60 S ribosomal subunits and eIF5 (Fig. 8, compare panels A,
B, and C). Furthermore,
[35S]Met-tRNAf bound to the 80 S initiation
complex reacted completely with puromycin to form methionyl puromycin
(Table I) indicating that the 80 S initiation complex
formed was functional in the peptidyl transfer reaction.
|
To determine whether eIF1A had any effect on the eIF5-mediated subunit joining reaction, isolated 40 S initiation complex containing bound [35S]Met-tRNAf was incubated with 60 S ribosomal subunits, limiting concentrations of eIF5 and increasing levels of eIF1A. As shown in Fig. 8, right panel, increasing levels of eIF1A had virtually no effect on the formation of the 80 S initiation complex. We conclude that eIF1A plays no role in the formation of the 80 S initiation complex.
Effect of eIF1A on the Association of Ribosomal SubunitseIF1A has been reported to bind to 40 S ribosomal
subunits and prevents the Mg2+-dependent
association of 40 S and 60 S ribosomal subunits (6). To investigate
whether eIF1A indeed functions as a ribosomal subunit anti-association
factor, we carried out the following series of reactions. First, we
incubated 40 S and 60 S ribosomal subunits in the presence of 1 mM MgCl2 with eIF1A and then added the
preformed [35S]Met-tRNAf·eIF2·GTP ternary
complex and AUG to the reaction. Formation of the 40 S initiation
complex was then analyzed by sucrose gradient sedimentation in the
presence of 1 mM MgCl2. As shown in Fig.
9, 40 S and 60 S ribosomal subunits, as expected, remained dissociated, and [35S]Met-tRNAf was
efficiently transferred to the 40 S ribosomes (see panels A
and E). At 1 mM MgCl2, ribosomal
subunits are known to remain dissociated in the absence of any added
protein (Ref. 31 and data not shown). If, however, the
[35S]Met-tRNAf·eIF2·GTP ternary complex
was added to ribosomal subunits that were preincubated at 5 mM MgCl2 in the absence of eIF1A, no
[35S]Met-tRNAf was transferred to the 40 S
ribosomes (Fig. 9, panel B). This is presumably because at
this elevated MgCl2 concentration, the ribosomal subunits
associated to form 80 S ribosomes (Fig. 9, panel F). If
eIF1A acts as a ribosomal subunit dissociation or anti-association
factor, the presence of eIF1A in reaction mixtures containing 40 S and
60 S ribosomal subunits should prevent 80 S ribosome formation, and
addition of [35S]Met-tRNAf·eIF2·GTP
ternary complex would lead to 40 S initiation complex formation.
However, as shown in Fig. 9, panels C and G, the
presence of eIF1A did not prevent association of ribosomal subunits to
form 80 S ribosomes, and in keeping with this no 40 S initiation
complex was formed. Under such conditions (5 mM
Mg2+ and with AUG codon), in the absence of 60 S
ribosomes, Met-tRNAf, as expected (see Fig. 1 and Refs. 13
and 15), was efficiently transferred to 40 S ribosomal subunits in an
eIF1A-independent manner (Fig. 9, panels D and
H). These results demonstrate that eIF1A, by itself, does
not act as a ribosomal subunit anti-association factor. The reasons for
the discrepancy between our results and those of Thomas et
al. (6) and Goumans et al. (32) which showed that eIF1A
acted as a ribosomal subunit anti-association factor are not completely
clear. It should be noted, however, that these investigators following
interaction of eIF1A with ribosomes added glutaraldehyde as a fixative
agent to reaction mixtures prior to sucrose gradient analysis. The use
of fixatives often introduces nonspecific binding interactions. Whether
eIF1A interacts with other proteins, e.g. eIF3 to cause
ribosome dissociation, remains to be investigated.
eIF1A was originally isolated from mammalian cells (33-35) and wheat germ extracts (30, 36) based on its ability to stimulate protein synthesis in a partially reconstituted system derived from rabbit reticulocyte lysates or wheat germ extracts. Purified eIF1A stimulated the translation of globin mRNA in the reticulocyte system 3-5-fold (33-35) and in the translation of plant viral mRNAs in wheat germ extracts nearly 10-fold (30, 36). This stimulatory effect of eIF1A in purified in vitro translation systems coupled with the recent demonstration (10) that the S. cerevisiae gene encoding eIF1A is essential for cell growth and viability is at variance with the weak effect eIF1A has for in vitro formation of 40 S and 80 S initiation complexes (6-9). It has been reported that in vitro, eIF1A stimulates both AUG-dependent or globin mRNA-dependent 40 S initiation complex formation about 1.5-2-fold by stabilizing the binding of Met-tRNAf to 40 S ribosomal subunits which facilitates subsequent mRNA binding (6-8). It has also been reported that eIF1A has a pronounced effect on the eIF5-mediated joining of 60 S ribosomal subunits to the 40 S initiation complex to form the 80 S initiation complex (7, 9). The factor has also been implicated in the dissociation of 80 S ribosomes to 40 S and 60 S ribosomal subunits (6, 32). Based on these observations, eIF1A has been designated a pleiotropic factor in the initiation pathway (4).
In the present work, we have used a model initiation system that directly measures the binding of Met-tRNAf (as Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomal subunit in the absence of mRNA to form the 40 S preinitiation complex. We have demonstrated that the transfer of Met-tRNAf (as Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomal subunit at physiological 1-2 mM Mg2+ requires the absolute participation of eIF1A. This effect did not require the presence of either mRNA or AUG in the reaction indicating that eIF1A is essential for the formation of 40 S preinitiation complex. In agreement with the results published from other laboratories (7-9), eIF3 also stimulated the transfer of the Met-tRNAf·eIF2·GTP ternary complex to 40 S ribosomes. However, the efficiency of the transfer reaction was much greater with eIF1A than with eIF3 alone (15-20-fold stimulation with eIF1A vis à vis 2-4-fold with eIF3). In the presence of eIF1A alone, the transfer of Met-tRNAf to 40 S ribosomes was nearly quantitative. Furthermore, under our conditions of analysis, we could not detect stable binding of eIF1A to the 40 S preinitiation complex thus convincingly demonstrating that the protein is not required to stabilize the binding of Met-tRNAf to 40 S ribosomes, as suggested by others (4-9). Additionally, the absence of eIF1A in the 40 S initiation complex isolated by sucrose gradient centrifugation has allowed us to use the complex as a direct substrate for the 60 S subunit joining reaction. We demonstrate that eIF5 alone was sufficient to mediate subunit joining reaction to form the 80 S initiation complex, and eIF1A had no effect on this reaction. The factor also has no demonstrable role in generating 40 S and 60 S ribosomal subunits from 80 S ribosomes.
Our results raise substantial doubt in the current view (4) that eIF1A is a pleiotropic factor in initiation of translation with only a marginal effect (1.5-2-fold) on 40 S initiation complex formation but with a somewhat greater effect on the subsequent joining reaction. The reasons for the discrepancy are not immediately apparent. The previous experiments (6-9) that led to the current model (4) of the initiation pathway measured 40 S initiation complex formation in in vitro reactions that included all known initiation factors. The function of a specific protein, e.g. eIF1A, was then determined by its omission from the complete system and measuring ribosomal initiation complex formation in its absence. An inherent problem with such experiments is that a trace contamination of the protein being analyzed in other initiation factor preparations would underestimate the requirement of this factor in initiation complex formation. This will be especially true for a low molecular weight protein like eIF1A which acts catalytically in the formation of 40 S preinitiation complex. Furthermore, the discrepancy between our finding that eIF1A does not directly affect subunit joining and those that show it does (7, 9) can be explained. Unlike our experimental approach, presented in Fig. 8, previous investigators did not utilize an isolated 40 S initiation complex, free of unreacted reaction components, as the substrate for the subunits' joining reaction. Rather, they formed the 40 S initiation complex in situ in the presence of either AUG codon (9) or globin mRNA (7) and all the required initiation factors except eIF1A and then added eIF1A, eIF5, and 60 S subunits to these reactions and measured the formation of 80 S initiation complex. Under these conditions, the observed increase of 80 S initiation complex formation by eIF1A can be accounted for by the stimulatory effect of this factor on the 40 S initiation complex formation.
Finally, it should be mentioned that although the present work demonstrates that eIF1A is essential to mediate nearly quantitative transfer of Met-tRNAf·eIF2·GTP ternary complex to free 40 S ribosomal subunits, it is likely that in vivo the Met-tRNAf·eIF2·GTP ternary complex binds, in the presence of eIF1A, to an eIF3·40 S complex rather than to free 40 S ribosomal subunits, since the majority of native 40 S subunits contains bound eIF3 (37, 38). We have, however, shown that the transfer of the ternary complex to 40 S subunits is inefficient in the presence of eIF3 alone, whereas eIF1A, by itself, is both necessary and sufficient to mediate highly efficient transfer of the ternary complex. These findings lead us to speculate that the major role of eIF3 is not in the initial transfer of the Met-tRNAf·eIF2·GTP ternary complex to 40 S ribosomes but rather in the subsequent binding of mRNA to the 40 S preinitiation complex. Similar conclusions regarding eIF3 were also reached by Trachsel et al. (7).
This paper is dedicated to the memory of Professor Julius Marmur. He was an outstanding scientist and an extraordinary human being who inspired us, by advice and example, to do research for the advancement of knowledge.
We are indebted to Dr. Jerard Hurwitz of the Sloan-Kettering Cancer Research Center, New York, for critically reading the manuscript. Mass spectrometric analysis was carried out in the Laboratory of Macromolecular Analysis of this institution under the guidance of Professor Ruth Angeletti.