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J. Biol. Chem., Vol. 275, Issue 23, 17740-17746, June 9, 2000
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From the Department of Chemistry, Hunter College and the Graduate
Center, City University of New York, New York, New York 10021
Received for publication, November 30, 1999, and in revised form, February 10, 2000
Recent studies demonstrated that wheat germ
poly(A)-binding protein (PABP) interacted with translation eukaryotic
initiation factor (eIF)-iso4G and eIF4B, and these interactions
increased the poly(A) binding activity of PABP (Le, H., Tanguay,
R. L., Balasta, M. L., Wei, C. C., Browning, K. S.,
Metz, A. M., Goss, D. J., and Gallie, D. R. (1997)
J. Biol. Chem. 272, 16247-16255) and the cap binding
activity of eIF-iso4F (Wei, C. C., Balasta, M. L., Ren, J.,
and Goss, D. J. (1998) Biochemistry 37, 1910-1916). We report here that the interaction between PABP and eIF-iso4G has a
substantial effect on the ATPase activity and RNA helicase activity of
(eIF4A + eIF4B + eIF-iso4F) complex. ATPase kinetic assays show, in the
presence of poly(U), PABP can increase the parameter
(kcat/Km) by 3.5-fold with
a 2-fold decrease of Km for the (eIF4A + eIF-iso4F)
complex. In the presence of globin messenger RNA, the ATPase activity
of the complex (eIF4A + eIF-iso4F) was increased 2-fold by the presence
of PABP. RNA helicase assays demonstrated that the presence of PABP
enhanced the RNA duplex unwinding activity of the initiation factor
complex. These results suggest that, in terms of the scanning model of translation initiation, PABP may enhance the mRNA scanning rate of
the complex formed by eIF4A, eIF4B, and eIF4F or eIF-(iso)4F and
increase the rate of translation.
Most eukaryotic mRNAs contain both a 5' cap
(m7GpppN) and a 3' poly(A) tail. The
cap-dependent binding of mRNA to the 40 S ribosome is
mediated by at least three eukaryotic initiation factors (eIF),1 eIF4F, eIF4A, and
eIF4B, and requires energy derived from ATP hydrolysis (1, 2). Early
studies have shown that the cap structure and poly(A) tail act in a
cooperative manner to regulate the translation of mRNAs (3, 4).
Recent studies indicated that the cooperative effect is related to the
interaction among eIF-iso4G, eIF4B, and PABP, which increases the
poly(A) binding affinity of PABP and the m7G-cap binding
affinity of eIF4F and eIF-iso4F (5, 6).
In wheat germ and other plants, an isozyme form of eIF4F called
eIF-iso4F has been found (7). eIF-iso4F shows an indistinguishable function from eIF4F in supporting in vitro translation (8). It contains two subunits, a 28-kDa eIF-iso4E and an 86-kDa eIF-iso4G. The eIF-iso4E acts as the binding site of m7GpppN cap. The
function of eIF-iso4G is not well characterized. It binds to mRNA
in an ATP-dependent manner, may interact with eIF4A and
eIF4B (9), and, more interestingly, may interact with PABP (5, 6).
cDNA analysis indicated that the amino acid sequence of eIF-iso4G
contained possible motifs for ATP binding, metal binding, and
phosphorylation (10). Both eIF4F and eIF-iso4F show
RNA-dependent ATPase activity, which is stimulated by the presence of eIF4A (11), and ATP-dependent helicase activity in the presence of eIF4A (12, 13). Recently, four initiation factors of
wheat germ, eIF4A, eIF4B, eIF-iso4E, and eIF-iso4G, were expressed in
Escherichia coli (14). In vitro translation measurements indicated that expressed initiation factors had the same
capacity to support translation in wheat germ extracts as native
factors purified from wheat germ (14).
In eukaryotic protein biosynthesis, the binding of mRNA to the
small (40 S) ribosomal subunit is the rate-limiting step and is a key
target for regulation (15-18). Two models were proposed for the
pathway of binding of mRNA to the 40 S ribosomal subunit. In the
first model (1), the first step in mRNA binding is the recognition
of m7GpppN cap by eIF4F or eIF-iso4F, and then unwinding of
secondary structure in combination with eIF4A and eIF4B in the
5'-untranslated region, to create a single-strand RNA, which serves as
the binding site for the 43 S preinitiation complex. In the second
model (19), eIF4E binds to m7GpppN cap first and then
associates with eIF4G, which is already bound to the 43 S complex.
Despite the differences in the two models, both models require that,
once bound to the cap, the eIF4F, eIF4A, and eIF4B complex must scan
along mRNA to unwind secondary structure before reaching the
initiation codon AUG. Therefore, the scanning step is most likely the
rate-limiting step for mRNA binding to the 43 S preinitiation
complex, and a potential target for regulation. The communication
between 5' cap and 3' poly(A) tail is a key regulation pathway for
mRNA translation (reviewed in Ref. 4). However, the role of PABP in
the scanning step has not been determined. In order to begin to answer
this question, in this investigation, we have studied the effect of
PABP on ATPase and RNA helicase activity of wheat germ initiation
factors. PABP stimulates the ATPase activity of initiation factors in a
poly(U)- or mRNA-dependent manner. The helicase
activity is also enhanced, suggesting a role for PABP in the rate of
scanning by the initiation factor complex.
Purification of Proteins--
eIF4A, eIF4B, eIF-iso4E, and
eIF-iso4G were expressed in E. coli containing the
constructed pET3d or pET23d (for eIF4A only) vector in BL21(DE3) pLyS
as described elsewhere (14). A HiTrap SP column from Amersham Pharmacia
Biotech was used to purify eIF-4B, eIF-iso4E, and eIF-iso4G by the
following procedure. E. coli cells were disrupted by
alumina, suspended in buffer B (20 mM HEPES/KOH, pH 7.6, 0.1 mM EDTA, 1.0 mM DTT, and 10% glycerol)
containing 600 mM KCl (B-600), and centrifuged at 45,000 rpm for 2 h. The supernatant was diluted with B-0 to a final
concentration of 60 mM KCl, and loaded onto a 2 × 5-ml Hitrap SP column and washed by B-60 buffer to base line. A
60-1000 mM KCl linear gradient of a total volume 80 ml was
used to elute the proteins. 1.0-ml fractions were collected and
analyzed by 10% SDS-gel electrophoresis. All steps were carried out in
a cold box at approximately 5 °C. The pH of buffer B used for
eIF-iso4E and eIF-iso4G purification was 7.0; for eIF4B it was 7.5. The
proteins appeared in the 200-300 mM KCl fractions. eIF4A
was purified by using a 10-ml column and His-Bind kits from Novagen.
The binding and washing buffer was 5.0 mM imidazole, 0.5 M NaCl, 20 mM Tris·HCl, pH 7.9. eIF4A was eluted by 100 mM imidazole, 0.5 M NaCl, 20 mM Tris·HCl, pH 7.9.
The purification procedure of PABP was as described previously (21)
with some modification. The 30-60% saturated ammonium sulfate
fraction of the 50-g wheat germ extract was loaded onto a 100-ml
Affi-Blue gel column. The column was washed with 4 M NaCl
in buffer A (10 mM HEPES/KOH, pH 7.6, 100 mM
KOAc, 1.0 mM CaCl2, 1.0 mM MgOAc,
1.0 mM phenylmethylsulfonyl fluoride, and 1.0 mM DTT), and PABP was eluted with 2.0 M
guanidine hydrochloride in buffer A. The eluted protein was dialyzed in
buffer A containing 10% glycerol, then 0.2 mg/ml poly(C) was added,
and the sample was loaded onto a 2.0-ml poly(A)-Sepharose 4B column,
and washed to base line by buffer A containing 0.5 M KCl.
PABP was eluted by 1 M urea and 2 M LiCl in
buffer A, and subsequently dialyzed against buffer A. Protein
concentrations were measured by the Bradford method, with bovine serum
albumin as a standard (22).
ATPase Assay--
The ATPase activity was determined by
measuring the release of 32Pi as described
previously (23). The standard assay was carried out in a reaction
volume of 20 µl, which contained 25 mM HEPES/KOH, pH 7.5, 100 mM KCl, 2.0 mM MgOAc, 1 mM DTT,
and 0.1 mM [ DNA and RNA Oligonucleotides--
DNA oligonucleotides were
synthesized by Genosys, and were purified by precipitation with 2 volumes of ethanol in TES (10 mM Tris, pH 7.5, 10 mM NaCl, 1.0 mM EDTA) adjusted to 0.5 M ammonium acetate, chilling at Transcription of Top Strand of RNA Duplex--
The top
strand of RNA duplex used in RNA helicase assay,
5'-GGGGAGA(A4C)5UAGCACCGUAAAGCACGC (a 50-mer
RNA oligonucleotide), was synthesized by in vitro
transcription using T7 RNA polymerase. The template for
transcription was composed of the following synthetic DNA
oligonucleotides: T7 polymerase promotor,
5'-GAATTTAATACGACTCACTATA, and
3'-CTTAAATTATGCTGAGTGATAT*CCCCTCT(TTTTG)5ATCGTGGCATTTCGTGCG-5' (* denotes the transcription start site). The transcription
reaction was performed using Ambion's MegashortscriptTM
transcription kit following the manufacturer's instructions. After the
reaction, the transcription solution was mixed with 10 M
urea, 0.01% bromphenol blue, and loaded on to a denaturing (8.0 M urea) 20% polyacrylamide (19:1 bisacrylamide:acrylamide) gel. The bands containing the transcription product were cut out and
extracted with 0.5 M ammonium acetate, 2.0 mM
EDTA, 0.1% SDS at 37 °C. Synthesized RNA oligonucleotide was
precipitated from the extraction buffer with 2.5 volumes of ethanol,
washed with ethanol, lyophilized, and resuspended in RNase-free water.
Concentrations of RNA oligonucleotides were measured by UV
spectroscopy. A value of 33 µg/1.0 A260 was
used to determine the concentration.
32P 5'-End-labeling of RNA Oligonucleotide--
The
bottom strand of RNA duplex (12-mer) was 32P-labeled at the
5'-end with T4 polynucleotide kinase from Amersham Pharmacia Biotech.
Forty picomoles of bottom strand RNA oligonucleotide and 10 pmol of
[ Helicase Substrate--
The RNA duplex used in the helicase
reaction was made by combining the top strand (50-mer) and
32P-labeled bottom strand (12-mer) oligonucleotides in a
1.5:1 ratio. The complementary strands were annealed (20 mM
Tris·HCl, pH 7.5, 80 mM KCl, 1.0 mM EDTA) in
a water bath heated to 98 °C for 5.0 min, and cooled slowly in a
cold box (about 5 °C) with gentle stirring. Under these conditions,
about 75% of the labeled oligonucleotide was hybridized.
Helicase Assay--
The assay was performed as described
previously (24) in a buffer that contained 20 mM HEPES/KOH,
pH 7.5, 70 mM KCl, 2.0 mM DTT, 2.0 mM magnesium acetate, and 2.0 units/µl RNase inhibitor RNAsin (Promega). The concentration of ATP was 2.0 mM,
duplex concentration was about 2.5 nM, and the
concentrations of eIF4A, eIF4B, eIF-iso4F, and PABP were 1.0 µM. The final reaction volume was 10 µl. The reaction
was started by addition of eIF4A or the initiation factor complex (as
indicated in Fig. 6), and incubated at 37 °C. Reactions were
terminated by adding 2.5 µl of a solution containing 50% glycerol,
2% SDS, 20 mM EDTA, and 0.01% bromphenol blue. Unwinding
reaction products were analyzed by separating the displaced labeled
bottom strand from the duplex by electrophoresis on 12% native
polyacrylamide gel in 1× TBE buffer at ambient temperature (about
25 °C). Radioautographs were generated by exposing the gels to Fuji
RX x-ray film and developed by standard procedures. The bands
containing the unwound labeled bottom strand and the duplex were cut
and counted. The percentage of unwound duplex was calculated by the
following formula: % unwound = (cpm of bottom band According to the scanning model of translation initiation, the
scanning step, i.e. scanning along mRNA and unwinding
secondary structure in the 5'-untranslated region of mRNA by the
complex formed by eIF4A, eIF4B, and eIF4F, requires energy derived from ATP hydrolysis (1, 19, 25). Recent studies have shown that, in the
wheat germ system, the communication between cap and poly(A) tail
involved the interaction between PABP and eIF-iso4G and eIF4B. The
interaction increased the poly(A) binding affinity of PABP and
m7GTP binding affinity of eIF-iso4F and eIF4F (5, 6). These results suggest PABP may also affect ATPase and helicase activity.
Fig. 1 shows the effect of PABP on the
ATPase activity of (eIF4A + eIF-iso4F) complex. Clearly, PABP
stimulates substantially the ATPase activity of the complex. The effect
of PABP on ATPase activities of initiation factores and their complexes
are summarized in Table I. The ATPase
activity of eIF4B or PABP alone, if any, is hardly detectable. eIF4A or
eIF-iso4G alone exhibits ATPase activity, consistent with previous
observations (11, 23). A mixture of equimolar concentrations of
eIF-iso4E and eIF-iso4G has the same ATPase activity as eIF-iso4G
alone. The ATPase activity of expressed eIF-iso4G or (eIF-iso4E + eIF-iso4G) is higher than eIF-iso4F purified directly from wheat germ
(11). In addition, from Table I we can see the ATPase activity of
(eIF4A + eIF-iso4E + eIF-iso4G) is higher than the additive ATPase
activity of eIF4A and (eIF-iso4E + eIF-iso4G) (Table I, 201 pmol > 26 + 91 pmol), indicating eIF4A has a synergistic stimulatory effect
on eIF-iso4F ATPase activity. However, the extent of stimulation of
eIF4A on ATPase activity of eIF-iso4F is much lower than its effect on eIF-iso4F purified from wheat germ (11). This may be due to the fact
that expressed eIF-iso4F is more active than the native eIF-iso4F
purified from wheat germ. One possible interpretation of this
difference is that the phosphorylation state of expressed eIF-iso4F is
different from that of native eIF-iso4F purified from wheat germ.
cDNA analysis indicated that eIF-iso4G has two potential protein
kinase C phosphorylation sites (amino acids 396-399 and 501-504) and
two potential casein kinase phosphorylation sites (amino acids 627-631
and 703-708) (10), both of which may be involved in translational
regulation. Phosphorylation of eIF4E has been shown to affect its
binding affinity for the cap structure (26). It was suggested that the
function of eIF-iso4F might be regulated by phosphorylation, which is a
post-translational phenomena (27). Despite the difference in ATPase
activity, expressed eIF-iso4F exhibited the same capacity to support
in vitro translation as native eIF-iso4F purified from wheat
germ when satellite tobacco necrosis viral RNA was used as the
messenger (14).
PABP exhibits a strong stimulation of ATPase activity of (eIF4A + eIF-iso4F) complex (Fig. 1 and Table I). The ATPase activity of (eIF4A + eIF-iso4E + eIF-iso4G + PABP) complex is 3 times the activity of the
complex without PABP (Table I, 647 pmol versus 201 pmol).
The presence of other initiation factors, such as eIF4B and eIF-iso4E,
has no substantial effect on the stimulatory effect of PABP (Table I).
However, 0.1 mM m7GTP (same as the
concentration of ATP in the assay solution) does produce inhibition of
the ATPase activity of (eIF4A + eIF-iso4E + eIF-iso4G) complex (Table
I, nos. 9 and 14). The inhibitory effect of m7GTP is
mediated by its interaction with eIF-iso4E, since no inhibition was
observed in the mixture without adding eIF-iso4E.
The stimulatory effect of PABP is shown more clearly in Fig.
2. The ATPase activity of eIF4A is
enhanced 2-fold by the presence of eIF4B, and is further stimulated by
the presence of PABP (Fig. 2A). The interaction between PABP
and eIF-iso4G greatly enhances the ATPase activity of the initiation
factor mixture (Fig. 2B). The ATPase activity of eIF-iso4G
alone is enhanced more than 2-fold by the presence of PABP (Table I,
nos. 15 and 16). But the ATPase activity of eIF-iso4G in the presence
of PABP accounts for less than one third of the ATPase activity of
(eIF4A + eIF-iso4G) or (eIF4A + eIF-iso4F) complex in the presence of
PABP (Table I, comparing no. 16 to no. 9 or 11). PABP enhances the
ATPase activity of (eIF4A + eIF-iso4F) by stimulating the synergistic
effect of the two factors.
Wheat Germ Poly(A)-binding Protein Increases the ATPase and
the RNA Helicase Activity of Translation Initiation Factors eIF4A,
eIF4B, and eIF-iso4F*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (total amount of ATP
was 2000 pmol; 200-800 cpm/pmol were used), 0.1 µg of poly(U), and
indicated amounts of initiation factors. For the ATPase kinetic assay,
5-µl aliquots of reaction mixture were taken from the standard assay
solution at the indicated times. The extent of hydrolysis of ATP was
measured within 5% of the initial ATP concentration in the kinetic
assays. All assays were performed at 25 °C. All values were
corrected for the amount of 32Pi released in
the absence of poly(U) and initiation factors.
20 °C for 2 h,
centrifuging and then washing with cold 70% ethanol. A 12-mer RNA
oligonucleotide (5'-GCUUUACGGUGC), the bottom strand of the RNA duplex
used in the helicase assay, was synthesized by Cybergen and purified by
a denaturing (8.0 M urea) 20% polyacrylamide (19:1
bisacrylamide:acrylamide) gel.
-32P]ATP (specific activity 3000 Ci/mmol, 10 mCi/ml)
were combined with 20 units of T4 polynucleotide kinase (20 µl final
volume), and the reaction was performed according to the
manufacturer's instructions. The labeled RNA oligonucleotide was
purified by a denaturing (8.0 M urea) 20% polyacrylamide
(19:1 bisacrylamide:acrylamide) gel as described above.
cpm of
background of bottom band)/(cpm of top band + cpm of bottom band cpm of background of bottom band). The cpm of the background of
bottom band was obtained from the corresponding band of the RNA duplex
in the absence of proteins (Fig. 6A, lane 1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (9K):
[in a new window]
Fig. 1.
Effect of PABP concentration on ATPase
activities of (eIF4A + eIF-iso4F) complex. The standard assay
solution (20 µl) contained 0.1 µg of poly(U), 3.0 µg of eIF4A,
2.5 µg of eIF-iso4E, 8.0 of µg eIF-iso4G, and indicated amount of
PABP.
ATPase activities of initiation factors and their complexes in the
standard assay solution containing 0.1 µg of poly(U)
). Variation among replicate
experiments was about 25% for different protein preparations; however,
the magnitude of the PABP stimulation varied less than 10% among
replicate experiments.
View larger version (14K):
[in a new window]
Fig. 2.
Stimulation of PABP on ATPase activities of
initiation factor complexes containing varying amount of eIF4A.
The standard assay solution (20 µl) contained 0.1 µg of poly(U),
indicated amounts of eIF4A, and the following amounts of initiation
factors where indicated, 6.0 µg of eIF4B, 2.5 µg of eIF-iso4E, 8.0 µg of eIF-iso4G, and 6.0 µg of PABP. In A and
B, filled circles refer to eIF4A
alone, open triangles refer to absence of PABP,
and open squares refer to presence of PABP;
A, (eIF4A + eIF4B); B, (eIF4A + eIF-iso4G).
The above results were obtained in the presence of poly(U). It is known
that the ATPase activity of the initiation factor complex is dependent
on the properties of the RNA present in the assay solution (11, 23). We
therefore investigated the effect of PABP on the ATPase activity of the
initiation factor complex in the presence of globin mRNA. Fig.
3 shows the effect of PABP on the ATPase
activity of (eIF4A + eIF-iso4F) complex in the presence of globin
messenger RNA. PABP increased the ATPase activity of the complex about
2-fold. However, the ATPase activity of the complex in the presence of
globin mRNA is about half the activity in the presence of
poly(U).
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An unexpected observation is that the stimulatory effect of PABP is
dependent on poly(U) concentration. Poly(U) at higher concentration
abolishes much of the effect of PABP on the ATPase activity of the
(eIF4A + eIF-iso4E + eIF-iso4G) mixture (Fig. 4A). In the absence of PABP,
increasing the poly(U) concentration leads to slight enhancement of
ATPase activity (Fig. 4B). It has been shown that (eIF4A + eIF-iso4F) or (eIF4A + eIF-iso4F) complexes have different ATPase
activity when different RNA were present in the assay solution (11,
23). The reason for this is not clear since the mechanism of (eIF4A + eIF4F) complex unwinding of RNA structure with energy derived from ATP
hydrolysis is not well understood. An interpretation of the effect of
poly(U) (shown in Fig. 4A) is that the inhibitory effect of
high poly(U) concentrations may be due to a mechanism whereby the
release of poly(U) is prior to the release of (ADP + Pi).
If poly(U) release is the rate-limiting step and is reversible, an
increase in poly(U) concentration would slow down the rate of the
reaction. Interestingly, such a mechanism would provide a motor force
for the movement along RNA.
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The parameters Km and
kcat/Km were determined for
the ATPase activity of (eIF4A + eIF-iso4E + eIF-iso4G) complex in the
presence or absence PABP (Fig. 5,
A and B). The Km value for the
mixture in the presence of PABP is 40 ± 5 µM; in
the absence of PABP, Km is 93 ± 22 µM. The presence of PABP induces a 2-fold decrease in
Km value. In addition, PABP leads to a 3-fold
increase in kcat/Km value.
There is no unique interpretation for the effect of PABP on the
Km or
kcat/Km value of (eIF4A + eIF-iso4F) complex since the ATPase activity of the complex is enhanced
synergistically by the presence of the both factors, while the
mechanism of this effect is unknown.
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The above results clearly demonstrate that PABP increased the ATPase
activity of the initiation factor complex. The effect of PABP on the
RNA helicase activity of the initiation factor complex therefore was
investigated. Fig. 6 shows the
autoradiography results of the RNA helicase assay. Fig. 6A
shows that PABP alone has no helicase activity. ATP hydrolysis is
required (lanes 5 and 6). Two
nonspecific RNA-binding proteins: MAX, which also binds DNA, and T7 RNA
polymerase, had no effect on the unwinding reaction (lanes
7-9). Wheat germ eIF4A has no significant unwinding activity of the RNA duplex (radiograph not shown; Fig. 6C).
eIF4B has some stimulation effect on the unwinding activity of eIF4A (Fig. 6C; radiograph not shown), whereas the same RNA duplex
can be unwound successfully by mammalian eIF4A and (eIF4A + eIF4B) complex (24). However, the (eIF4A + eIF4B + eIF-iso4F) complex exhibits
full RNA helicase activity of the RNA duplex (Fig. 6B, lanes 1-5). The RNA helicase activity of the
complex is ATP-dependent. In the absence of ATP, no
detectable unwinding of the RNA duplex is observed for the (eIF4A + eIF4B + eIF4-iso4F) complex (Fig. 6A, lane
5; data not shown). Additionally, PABP increases the RNA
duplex unwinding activity of (eIF4A + eIF4B + eIF-iso4F) complex (Fig.
6, b (lanes 6-10) and c).
These results suggest that the RNA helicase activity of the (eIF4A + eIF4B + eIF-iso4F) complex is coupled with the ATPase activity of the
complex.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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PABPs have been found in all eukaryotic cells studied thus far. Sequence analysis has revealed the extensive evolutionary conservation of the yeast, plant, and human PABP-encoding genes (20, 21). Early studies have suggested that PABP may be the mediator of the role of poly(A) tail (reviewed in Ref. 3). This suggestion has been confirmed by recent studies, which demonstrated that interactions among eIF4G, eIF4B, and PABP enhance the cap binding affinity of eIF-iso4F and poly(A) binding affinity of PABP (5, 6). These observations suggest that the interaction of PABP with eIF4G and eIF4B can increase the ability of the initiation factor complex to discriminate between capped and polyadenylated mRNA from capped nonpolyadenylated mRNA, or uncapped and polyadenylated mRNA, and therefore provide a competitive advantage for the efficient translation of capped and polyadenylated mRNA. Mutagenesis studies in the yeast system demonstrated that the interaction between eIF4G and PABP is a prerequisite for the stimulatory effect of poly(A) tail and the synergistic stimulatory effect of cap and poly(A) tail on in vitro translation (4, 28-30). These observations indicate that PABP is involved in translation initiation as part of the initiation factor complex.
In terms of the scanning model, the initial recognition of cap and poly(A) tail by the eIF4E and PABP in the initiation factor complex is the first step for the binding of 40 S ribosome to mRNA, which is followed by the unwinding of secondary structure in the 5'-untranslated region of mRNA with the energy derived from the hydrolysis of ATP (1, 19, 25). The previous studies indicated that the interaction between PABP and eIF4G enhances the initial recognition of cap and poly(A) tail by eIF4E and PABP (5, 6). Our present results show that the interaction between PABP and eIF4G and eIF4B also promotes the unwinding of secondary structure in the 5'-untranslated region of mRNA by the (eIF4A + eIF4B + eIF-iso4F) complex. In the scanning model of translation initiation, it is likely the scanning step, which seems to be a multiple step process (24), is the rate-limiting step of translation initiation, and a key target for translation regulation. The present results demonstrate that the interaction between PABP and eIF-iso4G increases both the ATPase activity and RNA helicase activity of the (eIF4A + eIF4B + eIF-iso4F) complex. Clearly, these results implicate PABP as an important component in the regulation of translation.
The mechanism of how the initiation factor complex unwinds RNA
secondary structure in the 5'-untranslated region of mRNA during translation initiation is not well understood. Our present results and
the results of previous assays demonstrate that the full RNA helicase
activity of eIF4A needs the concerted action of two other factors,
eIF4B and eIF4F or eIF-iso4F (24, 12). It has been suggested recently
that eIF4A might undergo a cycle of conformational changes during ATP
hydrolysis and that such conformational changes might be used by eIF4A
to transduce the energy derived from ATP hydrolysis to physical work to
unwind RNA secondary structure (31). Since there is no direct
interaction between PABP and eIF4A (5), the role of PABP in the RNA
helicase activity might be to stimulate the actions of eIF4B and eIF4F
or eIF-iso4F in promoting the conformational change of eIF4A. Another
possibility, suggested by the concentration dependence of poly(U)
stimulation of ATPase activity (Fig. 4), is that PABP is involved in
the release step of mRNA binding, which allows movement along the
mRNA. A simple mechanism would involve binding of eIF4A to
mRNA, stimulated by the presence of eIF4F or eIF-iso4F. eIF4B
subsequently stimulated the ATPase activity by increasing the release
of ADP, possibly through a cycle of conformational changes. PABP,
interacting with eIF4G, stabilizes the complex and allows movement
along the mRNA by promoting release from one binding site, which
has now become single-stranded, and progression further along the
mRNA to continue unwinding activity.
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ACKNOWLEDGEMENT |
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We thank Dr. Karen Browning for the plasmids containing the eIF clones and globin mRNA.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB-9722907 (to D. J. G.) and a Professional Staff Congress (PSC) City University of New York faculty award (to D. J. G.); Research Center in Minority Institution Award RR-03037 from the National Center for Research Resources of the National Institutes of Health supports infrastructure at Hunter College.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.
To whom correspondence should be addressed: Dept. of Chemistry,
Hunter College, City University of New York, 695 Park Ave., New York,
NY 10021. Tel.: 212-772-5383; Fax: 212-772-5332; E-mail: dgoss@
hejira.hunter.cuny.edu.
Published, JBC Papers in Press, March 15, 2000, DOI 10.1074/jbc.M909464199
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ABBREVIATIONS |
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The abbreviations used are: eIF, eukaryotic initiation factor; PABP, poly(A)-binding protein; m7GTP, 7-methylguanosine triphosphate; DTT, dithiothreitol.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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