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
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ABSTRACT |
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We have used an in vitro translation
initiation assay to investigate the requirements for the efficient
transfer of Met-tRNAf (as
Met-tRNAf·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. We observed that the 17-kDa initiation factor
eIF1A is necessary and sufficient to mediate nearly quantitative
transfer of Met-tRNAf to isolated 40 S ribosomal subunits.
However, the addition of 60 S ribosomal subunits to the 40 S
preinitiation complex formed under these conditions disrupted the 40 S
complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction
was carried out with 40 S ribosomal subunits that had been preincubated
with eIF3, the 40 S preinitiation complex formed included bound eIF3
(40 S·eIF3·Met-tRNAf·eIF2·GTP). In contrast to the
complex lacking eIF3, this complex was not disrupted by the addition of
60 S ribosomal subunits. These results suggest that in
vivo, both eIF1A and eIF3 are required to form a stable 40 S
preinitiation complex, eIF1A catalyzing the transfer of
Met-tRNAf·eIF2·GTP to 40 S subunits, and eIF3
stabilizing the resulting complex and preventing its disruption by 60 S
ribosomal subunits.
Initiation of translation in eukaryotic cells occurs by a sequence
of partial reactions requiring the participation of a large number of
specific proteins called eukaryotic translation initiation factors
(eIFs).1 In vitro
studies using purified initiation factors have shown that the overall
initiation reaction commences with the binding of the initiator
Met-tRNAf as the Met-tRNAf ·eIF2·GTP
ternary complex to a 40 S ribosomal subunit containing bound initiation factor eIF3 to form the 40 S preinitiation complex (40 S·eIF3·Met-tRNAf·eIF2·GTP). In the next step, the
40 S preinitiation complex binds to the capped 5'-end of mRNA in a
reaction requiring initiation factors eIF4F, eIF4A, and eIF4B and then
scans the mRNA to locate the correct initiation AUG codon where the
preinitiation complex is positioned on the mRNA to form the 40 S
initiation complex (40 S·eIF3·mRNA·Met-tRNAf·eIF2·GTP).
Subsequently, the 60 S ribosomal subunit joins the 40 S complex in an
eIF5-dependent reaction to form a functional 80 S
initiation complex (80 S·mRNA·Met-tRNAf; for
reviews, see Refs. 1-3).
We have investigated each of these partial reactions to define their
requirements. Recently, we described (4) an efficient in
vitro translation initiation assay in which the 17-kDa initiation factor eIF1A is necessary and sufficient for the transfer of the initiator Met-tRNAf (as Met-tRNAf·eIF2·GTP
ternary complex) to isolated 40 S ribosomal subunits in the absence of
mRNA (or an AUG codon) to form the 40 S preinitiation complex (40 S·Met-tRNAf·eIF2·GTP). Several laboratories have,
however, reported that the multimeric initiation factor eIF3 that binds
to the 40 S ribosomal subunit in vitro in the absence of
other initiation components (5) also stimulated the transfer of the
Met-tRNAf·eIF2·GTP ternary complex to 40 S subunits to
form the 40 S preinitiation complex (40 S·eIF3·Met-tRNAf·eIF2·GTP) (6-9). There is also
evidence that in vivo, the majority of native 40 S ribosomal
subunits contain bound eIF3 (10, 11). Thus, the possibility exists that
in vivo, in the presence of eIF1A, the
Met-tRNAf·eIF2·GTP ternary complex binds to a 40 S·eIF3 complex rather than to free 40 S ribosomal subunits to form
the 40 S preinitiation complex.
In this paper, we investigate the role of both eIF1A and eIF3 in the
formation of the 40 S preinitiation complex in vitro. We
show that these two proteins perform distinct functions in 40 S
preinitiation complex formation. Whereas eIF1A alone is necessary and
sufficient for the quantitative transfer of
Met-tRNAf·eIF2·GTP to isolated 40 S ribosomal subunits,
in reaction mixtures containing both 40 S and 60 S ribosomal subunits,
eIF3, in addition to eIF1A, is required to form a stable 40 S
preinitiation complex.
tRNA, Ribosomal Subunits, Purified Proteins, and
Antibodies--
The preparation of 35S-labeled rabbit
liver Met-tRNAf (20,000-100,000 cpm/pmol) and ribosomal
subunits from Artemia salina eggs was described previously
(12, 13). Initiation factor eIF2 was purified from rabbit reticulocyte
lysates by established procedures (4); homogeneous bacterially
expressed recombinant human eIF1A was isolated as described (4).
Purified eIF3 was isolated from rabbit reticulocyte lysates as we
described recently (9). It consists of six major polypeptides of 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa polypeptide reported
by others (14-17) to be a constituent of the eIF3 complex. These eIF3
preparations are highly active in forming AUG-dependent 40 S initiation complex that is fully competent in joining 60 S ribosomal
subunits to form a functional 80 S initiation complex (9). Rabbit IgG
antibodies specific for mammalian eIF1A were obtained as described (4), and total IgY antibodies specific for mammalian eIF3 subunits were
isolated from egg yolks of laying hens immunized with purified rabbit
reticulocyte eIF3 (9). Immunoblot analysis was as described previously
(4, 9).
Assay for Formation of the 40 S Preinitiation
Complex--
Formation of the 40 S preinitiation complex was measured
by eIF1A-dependent transfer of Met-tRNAf (as
the Met-tRNAf·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 (50 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, 6 µM GTP, 8 pmol of [35S]Met-tRNAf (20,000-50,000 cpm/pmol), and
0.8-1.2 µg of purified rabbit reticulocyte eIF2 were incubated at
37 °C for 4 min to promote formation of the
[35S]Met-tRNAf·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 as described previously (12). In stage 2, another set of
reaction mixtures (25 µl each) containing 20 mM Tris-HCl,
pH 7.5, 100 mM KCl, 2.5 mM 2-mercaptoethanol, 1 mM MgCl2, 0.6 A260 unit
of 40 S ribosomal subunits, and 30-100 ng of purified recombinant
eIF1A were incubated at 37 °C for 4 min and then chilled in an
ice-water bath. Each mixture was then supplemented with 45 µl of the
incubated stage 1 reaction mixture containing about 2 pmol of
[35S]Met-tRNAf·eIF2·GTP ternary complex.
After incubation at 37 °C for 4 min, reaction mixtures (now 70 µl
each) were chilled in an ice-water bath, the MgCl2
concentration of each reaction was adjusted to 5 mM, and
the mixture was layered onto a 5-ml 7.5-30% (w/v) sucrose gradient
containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 100 mM KCl, 5 mM
2-mercaptoethanol and centrifuged at 48,000 rpm for 105 min in an SW
50.1 rotor. Fractions (200-300 µl) were collected from the bottom of
each tube, and the radioactivity was determined in a liquid
scintillation spectrometer. Under the conditions of this assay,
formation of the 40 S preinitiation complex was totally dependent on
the presence of eIF1A, 40 S subunits, as well as the
[35S]Met-tRNAf·eIF2·GTP formed in stage 1 incubation. The efficiency of the 40 S preinitiation complex formation
was calculated relative to
[35S]Met-tRNAf·eIF2·GTP ternary complex
formed in stage 1 incubation.
Isolation of the 40 S Preinitiation Complex--
A reaction
mixture (175 µl) containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM 2-mercaptoethanol, 15 µg of
bovine serum albumin, 40 pmol of
[35S]Met-tRNAf (100,000 cpm/pmol), 18 µM GTP, and 12 µg of eIF2 was incubated for 4 min at
37 °C to form the
[35S]Met-tRNAf·eIF2·GTP ternary complex.
The reaction mixture was then supplemented with 1.8 A260 units of 40 S ribosomal subunits, 0.6 µg
of purified recombinant eIF1A, and MgCl2 (1 mM
final concentration). After incubation at 37 °C for 4 min, the
reaction mixture was chilled in an ice-water bath, and the
MgCl2 concentration was raised to 5 mM and then
resolved on sucrose gradients exactly as described above. Fractions
containing the 40 S preinitiation complex with bound
[35S]Met-tRNAf, free of unreacted reaction
components, were pooled, divided into small aliquots, and stored at
Formation of the 40 S Preinitiation Complex: Requirements for the
Binding of the Initiator Met-tRNAf to 40 S Ribosomal
Subunits in the Absence of mRNA--
We have shown previously (4)
that the 17 kDa-initiation factor eIF1A was both necessary and
sufficient for the transfer of Met-tRNAf to 40 S ribosomal
subunits (4). Whereas the multimeric initiation factor eIF3, which
binds to 40 S ribosomal subunits in the absence of other factors (5),
also can promote this transfer reaction, the efficiency of
Met-tRNAf transferred by eIF1A was far greater than with an
excess of eIF3 alone (4). Furthermore, the catalytic reutilization of
eIF1A in the transfer reaction was independent of eIF3. It was not
immediately apparent that the binding of eIF3 to 40 S ribosomal
subunits played any role in the formation of the 40 S preinitiation complex.
The reactions described above were performed with isolated 40 S
ribosomal subunits. To investigate complex formation under more
physiological conditions, namely when both 40 S and 60 S ribosomal
subunits were present in the same reaction, a preformed [35S]Met-tRNAf·eIF2·GTP ternary complex
was incubated with 40 S ribosomal subunits and eIF1A in the presence of
60 S ribosomal subunits, and the reaction products were analyzed by
sucrose gradient centrifugation (Fig. 1).
Although eIF1A, as expected, mediated the efficient transfer of
[35S]Met-tRNAf to 40 S ribosomal subunits to
form the 40 S preinitiation complex, the presence of 60 S ribosomal
subunits severely inhibited the formation of the 40 S preinitiation
complex (Fig. 1A). The addition of 60 S subunits reduced the
amount of Met-tRNAf bound to 40 S subunits from 1.2 pmol to
about 0.02 pmol (Fig. 1A). If however, the 40 S ribosomal
subunits were preincubated with both eIF1A and eIF3, the 40 S
preinitiation complex was formed efficiently even in the presence of 60 S ribosomal subunits (Fig. 1B). About 1.8 pmol of
Met-tRNAf was bound to 40 S ribosomes in the absence of 60 S ribosomal subunits; the addition of 60 S subunits to the preinitiation reaction resulted in the binding of nearly 1.4 pmol of
Met-tRNAf to 40 S ribosomal subunits. The efficient
formation of the 40 S preinitiation complex under these conditions
(i.e. in the presence eIF3 and 60 S ribosomal subunits) was
still dependent on the presence of eIF1A in the reaction mixture.
Omission of eIF1A from a preinitiation reaction containing only eIF3
resulted in a marked decrease in the amount of Met-tRNAf
bound to 40 S ribosomes (Fig. 1B). However, even under these
conditions, the small amount of the 40 S preinitiation formed in the
presence of eIF3 alone was unaffected by the addition of 60 S ribosomal subunits (Fig. 1B). These results demonstrate that in the
presence of 60 S ribosomal subunits, efficient formation of the 40 S
preinitiation complex requires the participation of both eIF3 and eIF1A
in the reaction. It should be noted here that in these 40 S
preinitiation reactions containing 60 S ribosomal subunits, no 80 S
initiation complexes were formed because initiation factor eIF5 was not
added to these reactions.
Role of eIF1A and eIF3 in the Formation of the 40 S Preinitiation
Complex--
The simplest interpretation of the results obtained above
is that the addition of 60 S subunits to 40 S preinitiation reactions at 5 mM MgCl2 resulted in the nonenzymatic
association of the ribosomal subunits to form 80 S ribosomes prior to
40 S preinitiation complex formation. It is well established that
formation of the 40 S preinitiation complex requires free 40 S
subunits; 80 S ribosomes cannot participate in this reaction.
Initiation factor eIF3 has been reported to bind to free 40 S ribosomal
subunits in the absence of initiator Met-tRNAf and other
initiation factors and prevent the
Mg2+-dependent association between 40 S and 60 S ribosomal subunits to form 80 S ribosomes (5, 10, 18, 19; also see
Ref. 3). Similar antiassociation activity has also been reported for
eIF1A (20). In light of this reported antiassociation activity of these
two initiation factors, the possibility exists that the presence of both eIF1A and eIF3 in the preinitiation reaction prevented association of the ribosomal subunits, thereby making free 40 S subunits available for 40 S preinitiation complex formation. We therefore investigated whether eIF3 and eIF1A indeed function as ribosomal subunit
antiassociation factors in the absence of
Met-tRNAf·eIF2·GTP ternary complex. Isolated 40 S
ribosomal subunits were incubated with eIF1A and eIF3 at 1 mM Mg2+; the Mg2+ concentration of
the reaction mixture was then raised to 5 mM, 60 S
ribosomal subunits were added, and the level of 80 S ribosome formation
was determined by sucrose gradient centrifugation (Fig. 2). In control incubations in which no
protein factors were added, ribosomal subunits, as expected, remained
dissociated at 1 mM Mg2+; at 5 mM
Mg2+, the ribosomal subunits associated to form 80 S
ribosomes (Fig. 2, left panel, A and
B). However, prior incubation of 40 S ribosomal subunits
with eIF3 and eIF1A followed by the addition of 60 S ribosomal subunits
failed to prevent association of the subunits to form 80 S ribosomes
(Fig. 2, left panel, C). Under similar assay
conditions, eIF6, which binds to 60 S ribosomal subunits, prevents the
association of 40 S and 60 S ribosomal subunits (data not shown here;
see Refs. 21-23). These results demonstrate that in contrast to other
reports (18-20), eIF1A and eIF3 by themselves do not have ribosomal
subunit antiassociation activity in the absence of
Met-tRNAf and other initiation components.
It should be noted that the lack of antiassociation factor activity of
eIF3 was not caused by the absence of binding of eIF3 to 40 S ribosomal
subunits. This was shown by incubating eIF3 with 40 S ribosomal
subunits followed by the addition of 60 S particles either at 1 mM Mg2+ (to keep the subunits dissociated) or
at 5 mM Mg2+ (to promote ribosomal subunit
association). In accord with a previous report (5), at 1 mM
Mg2+ when the ribosomal subunits remained dissociated, eIF3
bound stably to 40 S ribosomal subunits (Fig. 2, right
immunoblot, panel A). However, at 5 mM
Mg2+, the presence of 60 S ribosomal subunits resulted in
the release of eIF3 from 40 S ribosomal subunits (Fig. 2, right
immunoblot, panel B) with the concomitant formation of 80 S
ribosomes (compare Fig. 2, A and C, of left
panel). The release of eIF3 from the 40 S subunits is not caused
by the elevated Mg2+ concentration alone, since in the
absence of 60 S ribosomal subunits, eIF3 remained bound to 40 S
particles even at 5 mM Mg2+ (data not shown).
These results suggest that although eIF3 binds stably to 40 S ribosomal
subunits in the absence of Met-tRNAf·eIF2·GTP ternary
complex, eIF3 alone or in the presence of eIF1A is devoid of ribosomal
subunit antiassociation factor activity. Thus, the formation of the 40 S preinitiation complex by eIF3 and eIF1A in the presence of 60 S
ribosomal subunits is not the result of any ribosomal subunit
antiassociation activity of these two initiation factors.
These results suggested that the inhibition of eIF1A-mediated 40 S
preinitiation complex formation by 60 S ribosomal subunits in the
absence of eIF3 could be caused by displacement of the 40 S subunits
from the 40 S preinitiation complex formed in an eIF1A-dependent reaction. To investigate this possibility,
a preformed [35S]Met-tRNAf·eIF2·GTP
ternary complex was incubated with 40 S ribosomal subunits in presence
of eIF1A to form the 40 S preinitiation complex that was detected by
sucrose gradient centrifugation (Fig.
3A). When such a preformed 40 S preinitiation complex was incubated with 60 S ribosomal subunits and
then subjected to sucrose gradient centrifugation, the amount of
Met-tRNAf bound to 40 S ribosomes was reduced markedly
(Fig. 3A). If, however, 40 S ribosomal subunits were first
incubated with eIF3 and then used to form an
eIF1A-dependent 40 S preinitiation complex, the subsequent
addition of 60 S ribosomal subunits did not destabilize the
preinitiation complex (Fig. 3A). Western blot analysis
(using anti-eIF3 and anti-eIF1A antibodies as probes) of the gradient
fractions derived from the reaction in which the 40 S preinitiation
complex was formed in the presence of eIF1A and eIF3 was carried also
out (Fig. 3B). As shown, eIF3 was detected in the region of
the gradient where the 40 S preinitiation complex also sedimented (Fig.
3B, top panel). In contrast, anti-eIF1A antibodies did not detect eIF1A in this region; eIF1A was detected near
the top of the gradient at a position expected for a 17 kDa-protein (Fig. 3B, lower panel). These results show that
although eIF1A is released from the 40 S subunit after formation of the
40 S preinitiation complex, eIF3 remains bound to the complex.
Furthermore, in contrast to the complex lacking eIF3, the eIF3-bound
complex was not disrupted by 60 S ribosomal subunits. It should be
noted that in data not presented here, we have shown that 60 S-mediated disruption of the 40 S preinitiation complex in the absence of eIF3
leads to the concomitant formation of 80 S particles devoid of bound
Met-tRNAf.
Further Characterization of the Requirements for the Stabilization
of the 40 S Preinitiation Complex--
The results described above
suggest that the 40 S preinitiation complex formed in an
eIF1A-dependent reaction was stable in the presence of 60 S
ribosomal subunits only when the initiation reaction also contained
eIF3. Because eIF1A and eIF3 were present in these initiation
reactions, it was unclear whether eIF1A, which is essential for the
formation of the 40 S preinitiation complex, also plays a cooperative
role with eIF3 in the stabilization of the 40 S preinitiation complex.
To investigate this possibility, a preformed
[35S]Met-tRNAf·eIF2·GTP ternary complex
was incubated with 40 S ribosomal subunits and eIF1A in the absence of
AUG. The preinitiation complex formed was then isolated free of
unreacted reaction components by sucrose gradient centrifugation (see
"Experimental Procedures"). Western blotting using anti-eIF1A
antibodies confirmed that the purified isolated 40 S preinitiation
complex was devoid of eIF1A (data not shown here; see Ref. 4). The
stability of such an isolated 40 S preinitiation complex was then
investigated (Fig. 4). When the isolated
40 S preinitiation complex was again subjected to sucrose gradient
centrifugation, most of the bound Met-tRNAf dissociated
from the 40 S ribosomal subunit (Fig. 4). Released [35S]Met-tRNAf sedimented near the top of the
gradient (data not shown in the
figure).2 These results
indicate that an isolated 40 S preinitiation complex, purified free of
unreacted reaction components, cannot sustain a second round of
sedimentation through sucrose gradients even in the absence of 60 S
ribosomal subunits, indicating an intrinsic instability of the 40 S
preinitiation complex. If, however, the isolated 40 S preinitiation
complex was incubated with eIF3 and then subjected to sucrose gradient
centrifugation, most (approximately 88%) of the Met-tRNAf
remained bound to the 40 S preinitiation complex (Fig. 4). eIF1A, by
itself, did not stabilize the isolated 40 S preinitiation complex, and
eIF1A did not increase eIF3-mediated stability of the preinitiation
complex (Fig. 4). Furthermore, when the isolated 40 S preinitiation
complex was first incubated with eIF3, a substantial fraction of the
resulting preinitiation complex was not destabilized by 60 S ribosomal
subunits (Fig. 4). Whereas only about 6% of the preformed 40 S
preinitiation complex could be recovered on sucrose gradient
centrifugation after incubation with 60 S ribosomal subunits in the
absence of eIF3, the presence of eIF3 allowed the recovery of nearly
54% of the input 40 S preinitiation complex after incubation with 60 S
ribosomal subunits (Fig. 4, inset). The eIF3-mediated
stabilization under these conditions was not significantly improved by
the addition of eIF1A in the reaction mixture (data not shown). These
results show that although eIF1A is essential for the formation of the 40 S preinitiation complex, the presence of eIF3 bound to the 40 S
ribosomes is required for the stability of the 40 S preinitiation complex. eIF1A plays no role in this stabilization effect.
The 60 S ribosomal subunit-mediated dissociation of
Met-tRNAf from the 40 S preinitiation complex, formed in
the absence of eIF3, occurred prior to its recognition of the AUG
codon. If the 40 S preinitiation complex was allowed to interact with
AUG to form an initiation complex (40 S·AUG·Met-tRNAf·eIF2·GTP), subsequent incubation
with 60 S ribosomal subunits did not cause dissociation of
Met-tRNAf even in the absence of eIF3 (Fig.
5). This experiment also indicates that
the 40 S preinitiation complex formed in the absence of eIF3 is an
unstable complex with a conformation distinct from that of the 40 S
initiation complex.
An obligatory intermediate step in translation initiation in
eukaryotic cells is the initial binding of Met-tRNAf to 40 S ribosomal subunits (in the absence of mRNA) to form the 40 S
preinitiation complex (1-3). 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 this paper, we have investigated the role of eIF1A and eIF3 in the
formation of the 40 S preinitiation complex. Results presented here
show that although eIF1A can mediate nearly quantitative transfer of
Met-tRNAf·eIF2·GTP ternary complex to free 40 S
ribosomal subunits, the addition of 60 S ribosomal subunits totally
abolished 40 S preinitiation complex formation. This was shown to be
due to disruption of the preformed 40 S preinitiation complex (40 S·Met-tRNAf ·eIF2·GTP) by 60 S ribosomal subunits. If
the eIF1A-mediated 40 S preinitiation complex was formed using 40 S
ribosomal subunits preincubated with eIF3, the resulting 40 S
preinitiation complex (40 S·eIF3·Met-tRNAf·eIF2·GTP) was resistant to the
disruptive action of 60 S ribosomal subunits. These results along with
the previous observations made by Thompson et al. (10) and
Smith and Henshaw (11), that the majority of native 40 S ribosomal subunits contain bound eIF3, suggest that in the presence of eIF1A, the
Met-tRNAf·eIF2·GTP ternary complex binds to a 40 S·eIF3 complex rather than to free 40 S ribosomal subunits. Thus,
in vivo, when 40 S and 60 S ribosomal subunits are present
in the same milieu, both eIF1A and eIF3 are required to form a stable
40 S preinitiation complex, eIF1A catalyzing the transfer of
Met-tRNAf·eIF2·GTP to 40 S ribosomal subunits, and eIF3
stabilizing the complex against 60 S-mediated disruption.
The recruitment of the 40 S ribosomal subunits containing bound
Met-tRNAf·eIF2·GTP ternary complex to 5'-cap structure
of eukaryotic mRNAs occurs by interaction between eIF3 bound to the 40 S subunit with the eIF4G subunit of initiation factor eIF4F that is
associated with the 5'-cap structure of mRNA (24, 25). In light of
this finding, it is tempting to speculate on the physiological significance of the binding of eIF3 to 40 S subunits prior to the
transfer of Met-tRNAf to form the 40 S preinitiation
complex. First, as demonstrated in this work, the 40 S preinitiation
complex formed in an eIF1A-dependent reaction in the
absence of eIF3 is intrinsically unstable, and disruption of the
complex by 60 S subunits is a reflection of this instability. This is
consistent with our observation that the 40 S preinitiation complex not
containing bound eIF3 and purified free of unreacted reaction
components, by sucrose gradient centrifugation, is unstable to a second
round of sucrose gradient centrifugation (Fig. 4). Second, the presence of eIF3 in the 40 S preinitiation complex is a stringent prerequisite for it to bind to mRNA. A 40 S preinitiation complex formed in the
absence of eIF3 is a "dead-end" complex because it cannot bind
mRNA, and the disruption by 60 S ribosomal subunits leads to the
release of bound initiation factors that can then participate in
effective initiation reactions. Thus, it can be argued that the 60 S
subunit has a "proofreading" role ensuring that only a bona
fide 40 S preinitiation complex, one with bound eIF3, and thus
cued to bind mRNA in the subsequent step, is formed stably during
translation initiation.
Our results also demonstrate that eIF3 and eIFlA do not prevent 40 S
and 60 S ribosomal subunit association in the absence of other
initiation components and may not be involved in the generation of free
ribosomal subunits. It is worth noting that electronmicrographic
studies of Srivastava et al. (26) showed that eIF3 binds to
a site on the 40 S particle which is oriented away from the interaction
site with the 60 S subunit. The ribosomal subunit antiassociation
activity of eIF3 and eIF1A reported by other laboratories (10, 18-20)
could be caused by the use of fixatives (e.g.
glutaraldehyde) to stabilize the 40 S·eIF3 complex which may lead to
nonphysiological interactions. The results presented here, however show
that eIF3 does have antiassociation factor activity in the context of
40 S preinitiation reactions. In the absence of eIF3, 60 S subunits can
displace the 40 S subunit present in the 40 S preinitiation complex and
associate with 40 S subunits to form 80 S ribosomes. The presence of
eIF3 bound to the 40 S preinitiation complex prevents 60 S subunits
from displacing 40 S subunits from the preinitiation complex. Thus,
under these conditions, 80 S ribosomes cannot be formed.
The eIF3 preparations used in the present study were purified based on
their ability to stimulate the AUG-dependent binding of
Met-tRNAf to 40 S ribosomal subunits to form the 40 S
initiation complex (9). The subunit composition of these eIF3
preparations is somewhat different from that reported by others (see
Ref. 3), notably the absence of p170 polypeptide in our purified eIF3
preparations (9). We demonstrated previously (9) that the p170
polypeptide is a "dissociable" subunit of mammalian eIF3. Thus
although the presence of this polypeptide in eIF3 may be required for
the 40 S preinitiation complex to bind mRNA, or for its ribosomal
subunit antiassociation activity in the absence of other initiation
components, this polypeptide is dispensable for reactions described in
this paper.
Pestova et al. (27) have reported recently that initiation
factors eIF1A and eIF1 acted synergistically to mediate the assembly of
the 40 S ribosomal initiation complex at the initiation codon. In light
of our studies (Ref. 4 and this work) demonstrating that eIF1A is
essential for the formation of the 40 S preinitiation complex, it is
possible that the role of eIF1A on AUG selection observed by a Pestova
et al. (27) may be the result of the effect of eIF1A on 40 S
preinitiation complex formation prior to its binding to mRNA.
Further experiments are clearly necessary to establish whether eIF1A,
in addition to its function in the formation of the 40 S preinitiation
complex, is also essential for the selection of the AUG codon on
mRNA by the 40 S preinitiation complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of 60 S ribosomal subunits and eIF3 on
eIF1A-dependent 40 S preinitiation complex formation.
Panel A, reaction mixtures (50 µl) containing buffer A (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 2.5 mM 2-mercaptoethanol, 1 mM MgCl2),
0.3 A260 unit of 40 S ribosomal subunits, and
where indicated 50 ng of purified recombinant eIF1A, were incubated at
37 °C for 5 min, and the mixtures were chilled in an ice-water bath.
Each mixture was supplemented with 45 µl of a preformed
[35S]Met-tRNAf·eIF2·GTP ternary complex
reaction mixture containing 1.5 pmol of bound
[35S]Met-tRNAf (30,000 cpm/pmol) (see
"Experimental Procedures"). To one of the reaction mixtures
containing eIF1A ( ), 0.6 A260 unit of 60 S
ribosomal subunits was added, and the Mg2+ concentration of
all reaction mixtures was raised to 5 mM. After an
additional incubation for 5 min at 37 °C, reaction mixtures were
chilled in an ice-water bath, and the formation of the 40 S
preinitiation complex was measured by sucrose gradient centrifugation
in buffers containing 5 mM Mg2+ as described
under "Experimental Procedures."
, no eIF1A added;
, eIF1A
added but no 60 S ribosomal subunits;
, eIF1A + 60 S ribosomal
subunits added. Panel B, four reaction mixtures were
prepared and incubated as described under panel A except
that all reaction mixtures contained 2.5 µg of eIF3 and, where
indicated, 50 ng of eIF1A during the first incubation. After the
addition of 45 µl of a preformed
[35S]Met-tRNAf·eIF2·GTP ternary complex
reaction mixture containing 2 pmol of bound
[35S]Met-tRNAf, one of the reaction mixtures
containing eIF3 alone (
) and the other containing both eIF3 and
eIF1A (
) were treated with 0.6 A260 unit of
60 S ribosomal subunits. The Mg2+ concentration of all
reaction mixtures was then raised to 5 mM. After incubation
at 37 °C for 5 min, the mixtures were chilled in an ice-water bath,
and the formation of the 40 S preinitiation complex was analyzed by
sucrose gradient centrifugation as described under "Experimental
Procedures."
, eIF3 alone, no 60 S subunits;
, eIF3 +60 S
subunits;
, eIF3 + eIF1A, no 60 S subunits;
, eIF3 + eIF1A + 60 S
subunits. In both panels, the ascending arrows
indicate 35S radioactivity recovered at the top of each
gradient tube representing unreacted free
[35S]Met-tRNAf used during the initial
ternary complex formation as well as any unreacted
[35S]Met-tRNA·eIF2·GTP ternary complex. The position
of sedimentation of the 40 S particle was determined in a parallel
reaction and is indicated.
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Fig. 2.
Effect of eIF3 and eIF1A on the association
of 40 S and 60 S ribosomal subunits. Left panel, Three
reactions mixtures (A, B, and C), each
of 125-µl volume and containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM dithiothreitol (reaction
buffer), 1 mM MgCl2, and 0.6 A260 unit of 40 S ribosomal subunits were
prepared. Reaction mixture C contained in addition 5 µg of
eIF3 and 0.3 µg of eIF1A. After incubation for 4 min at 37 °C, 1.2 A260 units of 60 S ribosomal subunits was added
to each reaction mixture, and the Mg2+ concentration of
reactions B and C was raised to 5 mM,
but that for reaction A was maintained at 1 mM.
All reaction mixtures were incubated for an additional 4 min at
37 °C, chilled, and then layered onto 7.5-30% sucrose gradients in
reaction buffers containing either 1 mM MgCl2
(for reaction A) or 5 mM MgCl2
(reactions B and C) and centrifuged at 48,000 rpm
for 105 min in an SW 50.1 rotor. Each gradient was then fractionated in
an ISCO gradient fractionator, and the absorbance profile at 254 nm was
monitored. The sedimentation positions of 40 S, 60 S, and 80 S
ribosomes are indicated. Right panel, binding of eIF3 to 40 S ribosomal subunits. Two reaction mixtures, A and
B (25 µl each), containing the reaction buffer, 1 mM MgCl2, and 0.6 A260
unit of 40 S ribosomal subunits, were incubated with 10 µg of
purified eIF3 for 4 min at 37 °C. Subsequently, 1.2 A260 units of 60 S ribosomal subunits was added
to each reaction, and the Mg2+ concentration of reaction
B was adjusted to 5 mM, but that for reaction
A was maintained at 1 mM. After an additional
incubation for 3 min at 37 °C, the chilled reaction mixtures were
centrifuged in sucrose gradients as described under the left
panel, except that the MgCl2 concentrations in the
gradient buffers in reactions A and B were 1 and
5 mM, respectively. Aliquots of each gradient fraction were
then assayed for eIF3 by immunoblot analysis using chicken anti-eIF3
antibodies as probes. A, 40 S + 60 S subunits + eIF3 at 1 mM MgCl2. B, 40 S + 60 S + eIF3 at 5 mM MgCl2. The positions of sedimentation of 80 S ribosomes, 40 S ribosomal subunits, and free eIF3 are
indicated.
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Fig. 3.
Role of eIF3 in stabilizing a preformed 40 S
preinitiation complex against the destabilizing effect of 60 S
ribosomal subunits. Panel A, The reactions were carried
out in two separate stages. In stage 1, four separate reaction
mixtures, each of 50-µl total volume and containing buffer A (see
legend to Fig. 1), 0.3 A260 unit of 40 S
ribosomal subunits, and 100 ng of purified eIF1A, were prepared. Two of
the reaction mixtures ( ,
) also contained 2.5 µg of eIF3; the
other two (
,
) lacked eIF3. After the addition of 45 µl of a
preformed ternary complex reaction mixture containing 2 pmol of
[35S]Met-tRNAf·eIF2·GTP ternary complex
to each reaction, all mixtures were incubated at 37 °C for 5 min to
preform the 40 S preinitiation complex. In stage 2, two of the reaction
mixtures, one containing only eIF1A (
) and the other containing both
eIF1A and eIF3 (
) were treated with 0.6 A260
unit of 60 S ribosomal subunits. After raising the Mg2+
concentration of all reaction mixtures to 5 mM, they were
incubated at 37 °C for 5 min, chilled in an ice-water bath, and the
amount of the 40 S preinitiation complex remaining was determined by
sucrose gradient centrifugation as described under "Experimental
Procedures." Panel B, aliquots (25 µl) of sucrose
gradient fractions of the reaction mixture containing both eIF1A and
eIF3 but no 60 S subunits (
) were subjected to separate Western blot
analysis using either anti-eIF3 or anti-eIF1A antibodies as probes. The
positions of migration of purified eIF3 and eIF1A, probed with specific
antibodies, are shown in the leftmost lane of each
gel.
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Fig. 4.
eIF3 stabilizes isolated 40 S preinitiation
complex. For these experiments, eIF1A-dependent 40 S
preinitiation complex was formed in the absence of eIF3 and isolated
free of reaction components by sucrose gradient centrifugation as
described under "Experimental Procedures." Aliquots of the pooled
40 S preinitiation complex (40 S·[35S]Met-tRNAf·eIF2·GTP) containing
1.5 pmol of bound [35S]Met-tRNAf were used in
reaction mixtures (50 µl) containing 20 mM Tris-HCl, pH
7.5, 5 mM MgCl2, 100 mM KCl, and 1 mM dithiothreitol. Each reaction mixture was treated with
various additions as follows: , no addition;
, 2.5 µg of eIF3;
×, 100 ng of eIF1A;
, 2.5 µg of eIF3 + 100 ng of eIF1A;
, 0.5 A260 unit of 60 S ribosomal subunits;
, 2.5 µg of eIF3 followed by 0.5 A260 unit of 60 S
ribosomal subunits. After incubation at 30 °C for 5 min, each
reaction mixture was sedimented through 5-30% sucrose gradients as
described under "Experimental Procedures." Fractions were collected
from the bottom of each tube, and 35S radioactivity was
determined. The amount of [35S]Met-tRNAf
bound to 40 S ribosomes under each condition of incubation was
calculated and tabulated in the inset as the percent of the
stable 40 S preinitiation complex remaining. In each case,
[35S]Met-tRNAf released from the 40 S subunit
because of destabilization of the 40 S preinitiation complex was
recovered near the top of the gradient (indicated by
arrows). These values are not shown in the figure.
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Fig. 5.
Recognition of the AUG codon stabilizes the
40 S preinitiation complex in the presence of 60 S ribosomal subunits
even in the absence of eIF3. Four separate reaction mixtures, each
of 25-µl volume and containing buffer A, 0.3 A260 unit of 40 S ribosomal subunits, and 50 ng
of purified eIF1A, were prepared. One of the reaction mixtures ( )
also contained 2.5 µg of eIF3. After the addition of 50 µl of
preformed [35S]Met-tRNAf·eIF2·GTP ternary
complex reaction mixture containing 3 pmol of bound
[35S]Met-tRNAf, all reaction mixtures were
incubated at 37 °C for 4 min to form the 40 S preinitiation complex.
Each reaction mixture containing preformed 40 S preinitiation complex
was treated with 0.05 A260 unit of AUG and 0.6 A260 unit of 60 S ribosomal subunits as
indicated below.
, preformed 40 S preinitiation complex without
further treatment;
, preformed 40 S preinitiation complex treated
with 60 S ribosomal subunits;
, preformed 40 S preinitiation complex
first incubated with AUG at 37 °C for 3 min and then 60 S subunits
added;
, preformed 40 S preinitiation complex formed in the presence
of both eIF1, and eIF3 was first incubated with AUG for 3 min at
37 °C followed by the addition of 60 S ribosomal subunits. After
raising the Mg2+ concentration of all reaction mixtures to
5 mM, they were incubated again for 4 min at 37 °C,
chilled in an ice-water bath, and then analyzed for
[35S]Met-tRNAf binding to 40 S ribosomes by
sucrose gradient centrifugation as described under "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Jerard Hurwitz and Dr. Stewart Shuman of Memorial Sloan-Kettering Cancer Center, New York, and Dr. Dennis Shields of this institution for critically reading the manuscript. We also thank Supratik Das and Amitabha Bandyopadhayay of this laboratory for considerable help during the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by Grant GM15399 from the National Institutes of Health and by Cancer Core Support Grant P30CA13330 from the NCI, National Institutes of Health.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: Center for Blood Research, Harvard Medical
School, 200 Longwood Ave., Boston, MA 02115.
§ Present address: Dept. of Biology, Brandeis University, Waltham, MA 02254.
¶ To whom correspondence should be addressed: Dept. of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Jack and Pearl Resnick Campus, 1300 Morris Park Ave., Bronx, NY 10461.
2 In a separate experiment, we observed that nearly 70-80% of the released 35S radioactivity was retained on nitrocellulose membrane filters, indicating that Met-tRNAf was released from the 40 S subunit as [35S]Met-tRNAf·eIF2·GTP ternary complex.
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ABBREVIATIONS |
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The abbreviation used is: eIF(s), eukaryotic (translation) initiation factor(s).
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REFERENCES |
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