From the Department of Biochemistry, University of California, Riverside, California 92521
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ABSTRACT |
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Higher plants have two complexes that bind the
m7G-cap structure of mRNA and mediate
interactions between mRNA and ribosomal subunits, designated eIF4F
and eIFiso4F. Both complexes contain a small subunit that binds the
5'-cap structure of mRNA, and a large subunit, eIF4G or eIFiso4G,
that binds other translation factors and RNA. Sequence-specific
proteases were used to cleave native cap-binding complexes into
structural domains, which were purified by affinity chromatography. We
show here that eIFiso4G contains a central protease-resistant domain
that binds specifically to nucleic acids. This domain spans
Gln170 to Glu443 and includes four of the six
homology blocks shared by eIFiso4G and eIF4G. A slightly shorter
overlapping sequence, from Gly202 to Lys445,
had no nucleic acid binding activity, indicating that the N-terminal end of the nucleic acid binding site lies within Gln170 to
Arg201. The binding of the central domain and native
eIFiso4F to RNA homopolymers and double- and single-stranded DNAs was
studied. Both molecules had highest affinity for poly(G) and recognized single- and double-stranded sequences.
The group 4 translation initiation factors,
eIF4A,1 B, E, and G, bind and
recruit mRNA to 43 S pre-initiation complexes for translation
(reviewed in Refs. 1 and 2). Association of eIF4A and eIF4B confers
ATPase, RNA helicase, and RNA annealing activities on the intact
complex (1-3) and is thought to be necessary for melting of RNA
secondary structure and ribosome scanning to the AUG start codon. eIF4E
binds to the 5'-cap structure of mRNAs and associates tightly with
eIF4G. These two factors together form the core of the mRNA-binding
complex, designated eIF4F. Higher plants have two such complexes:
eIF4F, which is homologous to the mammalian complex, and eIFiso4F,
which is ~10-fold more abundant (4, 5). eIF4G and eIFiso4G differ
~2-fold in molecular mass and show only 25-30% sequence similarity
overall, with ~50% similarity in six small homologous regions (6).
Yet, despite this substantial divergence, these two factors have very
similar functions (1). Either factor alone is sufficient for
cap-dependent initiation in vitro (7, 8).
eIFiso4G and eIFiso4E have been cloned and sequenced from wheat and
have deduced molecular masses of 86.5 and 23.5 kDa, respectively
(6).
The eIF4G and eIFiso4G subunits of the mRNA-binding complexes act
as a central scaffold for binding of other translation factors, proteins, co-factors, and RNAs. These include binding sites within mammalian eIF4G for eIF4E (aa 409-457) (9), eIF3 (aa 480-886) (10),
and two sites for eIF4A (aa 887-1402 and 478-883) (10, 11). Plant
eIFiso4G also binds eIFiso4E near its N terminus (aa 52-90) (12) in a
small region with sequence homology to the mammalian factor. Binding of
eIF4G and eIFiso4G to RNAs, distinct from the cap recognition function
of the intact complex, has been demonstrated by several laboratories
and appears to play a critical role in the cap-independent initiation
of viral RNAs. Sha et al. (13) calculated the equilibrium
binding constants of recombinant wheat eIFiso4G for both capped and
uncapped mRNAs. They found that the binding constants for uncapped
message were comparable to those of eIFiso4E and eIFiso4F for capped
mRNA, clearly indicating that the large subunit also binds mRNA
efficiently and that it does not discriminate between capped and
uncapped messages. These findings are consistent with the role of eIF4G
in the cap-independent translation of viral mRNAs, following
cleavage and loss of the eIF4E binding domain by viral proteases (10,
14). In functional assays for initiation complex formation, Pestova
et al. (15) have shown that only eIF4A and the central third
of human eIF4G (aa 457-932) are required for binding of
encephalomyocarditis virus RNA to 43 S ribosomal complexes, and that
this central eIF4G peptide binds, and cross-links to, the viral
internal ribosome entry site (IRES). Wheat eIF4F and eIFiso4F were also
reported to bind poly(A) mRNA (16), which implies that the
cap-binding complex interacts with both the 5' and 3' termini of intact
mRNAs, perhaps ensuring that only intact messages are translated.
The binding of eIFiso4G to other proteins, in addition to translation factors, has also been reported. The N-terminal region of eIFiso4G appears to bind poly(A)-binding protein (17), thereby increasing its
activity. Additionally, recombinant wheat eIFiso4G binds to, and
promotes the bundling of microtubules in vitro, and
co-localizes with microtubules in vivo, suggesting a role
for eIFiso4F in localizing the translational machinery to the
cytoskeleton (18). The specific sites and structural motifs in eIFiso4G
or eIF4G that mediate most of these RNA and protein interactions have
not yet been identified.
In this study, we have used limited proteolysis and affinity
chromatography to isolate functional domains of the mRNA-binding complexes of wheat. Protease-resistant domains were fractionated by
affinity chromatography on phosphocellulose (phosphate interactions), oligo(dT)-cellulose (phosphodiester or base interactions), and m7GTP-sepharose (cap recognition). Peptides eluted from the
different affinity columns were purified by reverse phase HPLC,
sequenced to determine their N terminus, sized by mass spectrometry,
and compared with published sequences of cloned factors to localize each peptide within the primary amino acid sequence. We report here the identification of a central protease-resistant domain in
eIFiso4G, which binds both nucleic acids and phosphocellulose. The specific affinities of both intact eIFiso4F and the central domain
of eIFiso4G were determined for RNA homopolymers and ss- and dsDNAs.
Reagents--
Sequencing grade trypsin and endoproteinase glu-C
were from Boehringer Mannheim. Oligo(dT)-cellulose and
m7GTP-Sepharose 4B were from Amersham Pharmacia Biotech.
Phosphocellulose was from Whatman. Agarose-conjugated poly(A), poly(C),
poly(G), poly(U), ssDNA, and dsDNA were from Sigma. Oligonucleotides
were from Sigma. All other chemicals were purchased from Sigma, U. S.
Biochemicals, Bio-Rad, or Fisher Scientific.
Protein Purification--
mRNA cap-binding complexes were
purified from wheat germ by a modification of the method of Lax
et al. (Ref. 19; note that eIFiso4G is designated eIF-4B in
this reference). Wheat germ (150 g) was ground with 10% each fine sand
and polyvinylpolypyrrolidone (20), homogenized in four volumes of iced
buffer (0.25 M sucrose, 200 mM Tris/Hepes, pH
8.6, 400 mM potassium acetate, 50 mM magnesium acetate, 5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.07 mg/ml soybean trypsin
inhibitor), and centrifuged 10 min at 10,000 × g. The
supernatant was decanted through 70 µm nylon mesh, centrifuged 2 h at 368,000 × g, precipitated with 70% ammonium
sulfate, and dialyzed against buffer B120 (buffer "B" contains 20 mM Hepes/KOH, pH 7.6, 1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerin, and the concentration of KCl, in
mM, indicated by the suffix). Material from five or six
preparations was adsorbed to a 50-ml m7GTP-Sepharose 4B
column, and mRNA cap-binding complexes were eluted with 100 µM m7GTP in B120. Approximately 20 mg of
protein was recovered and applied to a 30-ml phosphocellulose column.
eIF4A did not bind, and eIFiso4F eluted with a linear salt gradient
(B80 to B1000) at approximately 150 mM KCl. Fractions
containing eIFiso4F were concentrated and desalted in Centricon-10 spin
cartridges (Amicon). Preparations of cap-binding complexes at this
stage contained both eIFiso4F and eIF4F, and were used to identify
peptides from both complexes which bound to affinity resins (Figs.
1-3). In replicates of these experiments and all subsequent binding
specificity studies (Figs. 5 and 6 and Tables I and II), eIFiso4F was
further purified by HPLC using a DEAE column (Waters AP Mini, 5 × 50 mm) eluted with a linear salt gradient (B80 to B500). At each step,
fractions were analyzed for protein content by Bradford assays or
spectrophotometry at 280 nm, and by SDS-PAGE (21) on 10% gels stained
with Coomassie Brilliant Blue R250 or silver (22).
Proteolytic Digestions--
Limited protease digestion under
native conditions was according to Geisler and Weber (23). Digestion
reactions contained 10 mM Hepes/KOH, pH 7.5, 50 mM KCl, 30% glycerol, 10 mM
Affinity Fractionation of Protein Digests--
For the results
reported in Fig. 2, 300-500 µg of digested cap-binding complexes
were chromatographed on 2-ml phosphocellulose, oligo(dT)-cellulose, or
m7GTP-Sepharose columns, and eluted with a 12-ml linear
salt gradient (B100 to B1000). All fractions were analyzed for protein
composition by SDS-PAGE on 14% gels with silver staining, and by
fluorescence spectroscopy at excitation and emission wavelengths of 280 and 350 nm, respectively. Only selected fractions are shown.
Binding studies with ribonucleotide homopolymer and ss and dsDNA resins
were carried out in spin columns using 20-100 µg of protein and 100 µl of packed resin in microcentrifuge filter cups (MSI, Westboro, MA;
0.45-µm acetate filters). Solutions (300-µl aliquots) were spun
through the resin at 500-1,000 rpm for 2-5 min; at least three washes
(nine column volumes) were used at each step. Columns were equilibrated
in B50 and eluted sequentially with 0.1, 1, or 4 mM NTPs or
dNTPs, 200 µM oligo(dT)6 or
oligo(dT)12, or with increased KCl, as indicated. All
fractions were analyzed as above, but only selected fractions are
shown. eIFiso4F, endoproteinase glu-C digests of eIFiso4F, and purified
E31 were chromatographed on all affinity resins at least twice.
Purification, Sequencing, and Mass Spectrometric Analysis of
Peptide Fragments--
Proteolytic cleavage products that bound
affinity resins were further purified by reverse phase HPLC on a C18
column (Waters, 3.9 × 150 mm) equilibrated in 0.1%
trifluoroacetic acid. Elution was with a linear acetonitrile gradient
(0-80%) containing 0.1% trifluoroacetic acid. Absorbance was
monitored at 205 nm. Peaks were concentrated in a Centrivap
concentrator and analyzed by SDS-PAGE and silver staining. The
N-terminal sequence of purified peptides was determined by Edman
degradation at the UCR Biotechnology Instrumentation Facility, or at
the Stanford Biotechnology Facility. Peptide masses were determined by
time of flight mass spectrometry by Curtis Monnig in the Department of
Chemistry at UCR, or at the Harvard Biotechnology Facility. The
locations of peptides within wheat eIFiso4G and eIFiso4E were
determined by comparison with published sequences (Ref. 6; GenBank
accession numbers M95747 and M95819, respectively). Other sequences
used for comparison were; maize
eIFiso4G,2 thale cress
eIFiso4G (AC005170),3 wheat
eIF4G,4 human eIF4G (Ref. 25;
D12686), rabbit eIF4G (Ref. 26; L22090),
yeast eIF4G (Ref. 27; L16923), and human eIF4G2 (Ref. 28; AF012072).
Sequence alignments were done using the BestFit program in GCG (29) and
refined by hand.
Identification of Protease-resistant Domains in Wheat mRNA
Cap-binding Complexes Which Bind Phosphocellulose,
Oligo(dT)-Cellulose, and
m7GTP-Sepharose--
Preparations of wheat
cap-binding complexes contain eIFiso4F, a small amount of eIF4F,
and a trace amount of eIF4A which is bound to eIF4F (Fig.
1 left), as determined by
immunoblotting (data not shown). These preparations were the starting
point for identifying peptides from either complex that bound to
affinity resins. Purified eIFiso4F (Fig. 1, left) was used
in the nucleic acid binding experiments described below.
Cap-binding complexes were cleaved into protease-resistant domains by
digestion with trypsin or endoproteinase glu-C under native conditions
(Fig. 1). Several large peptides were reproducibly generated that
withstood even prolonged digestion. After 16 h of incubation with
trypsin, two main bands remained, which migrated on SDS-PAGE with
apparent molecular masses of 28 and 26 kDa. Minor bands between 20 and
24 kDa were more abundant in some preparations. The breakdown pattern
generated by endoproteinase glu-C was more complex; approximately 10 protease-resistant bands, with apparent molecular masses between 14 and
31 kDa, were generated in a 9-h digest. Digestion times of 12 h
for trypsin and 8 h for endoproteinase glu-C were used in the
experiments shown below.
In order to identify and purify peptides with specific binding
affinities, digests of cap-binding complexes were chromatographed in
parallel on phosphocellulose, oligo(dT)-cellulose, and
m7GTP-Sepharose. When trypsin digests were applied to
phosphocellulose only T28, the largest stable fragment, bound and was
eluted from the column in ~150 mM KCl (Fig.
2 top left, lane
4). In contrast, none of the peptides generated by trypsin
digestion bound to oligo(dT)-cellulose (Fig. 2 bottom left).
The second major band at ~26 kDa was primarily undigested eIFiso4E,
which was resistant to trypsin digestion for up to ~16 h. It bound to
m7GTP-Sepharose (data not shown) and had no affinity for
either phosphocellulose or oligo(dT)-cellulose (Fig. 2). This binding pattern was the same as for native eIFiso4E (data not shown).
Cap-binding proteins digested with endoproteinase glu-C were
chromatographed as above (Fig. 2 right). Major bands with
apparent masses of 31, 23, and 22 kDa were retained on
phosphocellulose, and eluted in 150-200 mM KCl (Fig. 2
top right, lanes 4 and 5). The two smaller bands were also retained on m7GTP-Sepharose
(data not shown), while the 31-kDa band bound to oligo(dT)-cellulose
(Fig. 2, bottom) and eluted at ~150 mM KCl. Further fractionation of the 31-kDa band by RP-HPLC revealed two peptides with very similar size and hydrophobicity, running as a barely
resolved doublet on SDS-PAGE, and eluting from a C18 column at only a
1% difference in acetonitrile concentration (Fig. 3). Only the larger of these two
peptides, corresponding to the second RP-HPLC peak, was present in
digests of purified eIFiso4F, and was designated E31.
E31 binding to phosphocellulose and oligo(dT)-cellulose was due to its
direct interaction with the resins, rather than to a protein:protein
interaction with the second peptide, or with one of the other peptides
retained on the columns. Purified E31, isolated from digests of
purified eIFiso4F by phosphocellulose chromatography and DEAE-HPLC, had
the same affinity and elution profile on phosphocellulose and
oligo(dT)-cellulose as did E31 in digests (data not shown).
Peptide Assignments--
Amino acid sequencing and mass
spectrometry were used to identify those peptides that bound to
affinity resins. Seven amino acid residues were sequenced from the N
terminus of each fragment; each was sequenced twice from separate
purifications, and E31 was sequenced following both phosphocellulose
and oligo(dT)-cellulose purification. The C-terminal residues were
calculated from the total mass and amino acid composition. T28 and E31
were found to be overlapping peptides from the central portion of
eIFiso4G with 242 amino acids, from Gly202 to
Glu443, in common (Table I).
The additional 32 amino acids at the N terminus of E31, from
Gln170 to Arg201 (Fig.
4A), are required for
oligo(dT)-cellulose binding activity. This sequence thus appears to
form part of a nucleic acid recognition site.
The second 31-kDa peptide, present in endoproteinase glu-C digests of
cap-binding complexes, originated from eIF4G. Its N-terminal sequence
was KKYVVGK, which is located 20 amino acids N-terminal to the second
region of homology shared by eIFiso4G and mammalian eIF4G (12),
beginning at Lys215 in eIFiso4G (Fig. 4A, Region
2). When the sequences of wheat eIF4G and eIFiso4G were compared
in this region, the start site of the eIF4G peptide was seen to lie
between those of E31 and T28, and to include only seven of the 32 additional residues present in E31 (Fig. 4A). There is
limited sequence conservation between factors and species in this
region, but several short conserved sequences and amino acids are
present in almost all of the factors sequenced to date (Fig.
4B). Most prominent are two small charge clusters,
corresponding to KAD194 and EKDR209 in
eIFiso4G, which flank conserved Trp, Ser, and Arg residues present at
the N terminus of the eIF4G peptide. These elements are also present in
yeast eIF4G, but are much more widely spaced (Fig. 4B).
Digestion with endoproteinase glu-C produced two peptides of ~22-23
kDa, which were retained on phosphocellulose (Fig. 2, top
right, lanes 4 and 5). These
bands were identified as eIFiso4E peptides by their affinity for
m7GTP-Sepharose (data not shown), their co-migration with
the two main fragments generated by digestion of purified eIFiso4E
(data not shown), their intense fluorescence (eIFiso4E has an unusually high tryptophan content), and by sequencing of the smaller peptide, which was designated E21. The N-terminal sequence of E21 was AKGPHKL and its mass was 20,852 Da, corresponding to
Ala29-Val209 of eIFiso4E (6). Truncation of
the N-terminal 28 amino acids did not alter the affinity of E21 for the
cap structure, as determined by fluorescence quenching analysis (data
not shown). This finding agrees with results reported by Chavan
et al. (30), who identified a trypsin-resistant core domain
in human eIF4E (aa 47-182), which also retained its affinity for
m7GTP. Native eIFiso4E does not bind either
phosphocellulose or oligo(dT)-cellulose (data not shown), but is
retained on both columns when bound to eIFiso4G. Retention of the
eIFiso4E peptides on phosphocellulose could thus have resulted from
their interaction with E31. This proved not to be the case, however, as
purified E21 alone bound to phosphocellulose (data not shown).
Apparently the loss of seven Asp and Glu residues from the N terminus
leaves several unpaired positive charges, which can interact with
phosphate groups.
Purified eIFiso4F and E31 Bind Specifically to Nucleic
Acids--
Intact eIFiso4F could not be eluted from
oligo(dT)-cellulose by free dNTPs, NTPs, or NMPs, even at
concentrations as high as 4 mM (data not shown), but were
readily eluted by 200 µM oligo(dT)12 (Fig.
5, top). As the concentration
of phosphate groups in 4 mM NTP is 5-fold higher than that
of the oligo solution, this interaction cannot be due simply to charge.
In addition, oligo(dT)6 failed to elute eIFiso4F (Fig. 5,
bottom), indicating that the minimum length of the target
sequence required for eIFiso4F binding was between 7 and 12 nucleotides.
The binding specificities of intact eIFiso4F and E31 were determined
for RNA homopolymers and random sequence ss- and dsDNAs (Table
II and Fig.
6). The binding specificity of purified
E31 was the same as for E31 in digests, and in each case where binding occurred, free nucleoside mono or triphosphates failed to elute eIFiso4F or E31 from the columns (Fig. 6 and data not shown). eIFiso4F
and E31 bound to poly(G) with the highest affinity, and showed no
interaction with poly(C). Unlike eIFiso4F, E31 showed no affinity for
poly(U) and weak affinity for poly(A), being retarded on
poly(A)-agarose but eluting in the wash fractions (data not shown).
T28, which did not bind oligo(dT) (Fig. 2), also did not bind poly(G)
(data not shown). These results indicate that the central domain of
eIFiso4G mediates binding of the eIFiso4F complex to poly(G),
oligo(dT), and phosphocellulose, and that this domain recognizes both
single- and double-stranded sequences. The lack of interaction of E31
with poly(A) and poly(U) suggests that these lower affinity binding
sites lie outside of the central domain. However, it is possible that
these nucleic acids are also bound by the central domain, but that
additional sequences located elsewhere in the molecule are required to
stabilize their interactions.
We have shown that wheat eIFiso4G contains an endoproteinase
glu-C-resistant structural core, which binds nucleic acids with base
specificity and poly(G) with highest affinity. This domain is centrally
located in eIFiso4G, and spans four of the six small regions of
homology it shares with mammalian eIF4G (Fig.
7) (1, 6). A corresponding domain from
eIF4G was also purified, and our results suggest that it has many of
the same properties; it was resistant to endoproteinase glu-C
digestion, bound oligo(dT)-cellulose and phosphocellulose, migrated
closely with E31 on SDS gels, and had very similar hydrophobicity. The
N-terminal end of the nucleic acid recognition site in eIFiso4G lies
within Gln170-Arg201, because the shorter
tryptic peptide (T28) lacks these 32 residues and fails to bind nucleic
acids. Only the later third of this sequence is conserved among eIF4G
factors, and only the last seven amino acids, corresponding to
Val195-Arg201 of eIFiso4G, are also present in
the eIF4G peptide (Fig. 4). This suggests that the N-terminal end of
the nucleic acid binding site lies within these seven residues, which
form part of an additional short block of homology centered at the N
terminus of the tryptic peptide (Figs. 4B and 7). This
sequence, from Lys192 to Arg209, is ~50%
similar in eIF4G isoforms from all species, a comparable level of
homology to the other six conserved regions (1). Further studies of
purified eIF4G are required to define structural and functional
properties of its central domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, cap-binding complexes or purified eIFiso4F (0.6 mg/ml for trypsin or 0.9-1.5 mg/ml for endoproteinase glu-C), and
either 1% trypsin or 2% endoproteinase glu-C (by mass, relative to
protein sample). Trypsin cleaves on the carboxyl side of arginine and
lysine residues, and endoproteinase glu-C cleaves on the carboxyl side
of aspartate and glutamate residues. Incubations were at room
temperature for the times indicated. Reactions were stopped by freezing
in liquid nitrogen, and the cleavage products analyzed on 14% gels by
SDS-PAGE and silver staining.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Proteolytic cleavage of cap-binding
complexes. Left panel, wheat germ cap-binding complexes
(CBC) were used directly, or further purified by DEAE-HPLC
fractionation to separate eIF4F (4F) from eIFiso4F
(i4F). Analysis was by SDS-PAGE on a 10% gel stained with
Coomassie Blue. Factor assignments are shown at left:
4G, eIF4G; i4G, eIFiso4G; 4A, eIF4A;
i4E, eIFiso4E; and 4E, eIF4E. Digestions of
cap-binding complexes with trypsin (center panel) or
endoproteinase glu-C (right panel) were for the hours
indicated. Products were analyzed by SDS-PAGE on 14% gels with silver
staining. Molecular masses of standard proteins in kDa are indicated to
the left.
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Fig. 2.
Affinity fractionation of peptides from
cap-binding complexes. Trypsin (left) and
endoproteinase glu-C (right) digests were chromatographed on
either phosphocellulose (top, PC) or
oligo(dT)-cellulose (bottom, dT). Elution
profiles were monitored by fluorescence. Insets, SDS-PAGE of
selected fractions, run on 14% gels and silver-stained.
i4F, eIFiso4F standard. T, trypsin digest of
cap-binding complexes. E, endoproteinase glu-C digest of
cap-binding complexes. Numbered lanes correspond
to labeled fractions in the elution profiles. The molecular masses of
standard proteins in kDa are indicated to the left of each
gel.
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Fig. 3.
Separation of E31 and a homologous eIF4G
domain by RP-HPLC. An endoproteinase glu-C digest of cap-binding
proteins was sequentially chromatographed over phosphocellulose and
oligo(dT)-cellulose to yield the 31-kDa band, which was further
fractionated by RP-HPLC. Only part of the 60-min RP-HPLC elution
profile is shown; no other peaks were detected. Inset,
SDS-PAGE of the initial endoproteinase glu-C digest (E), the
31-kDa band (31), and the two RP-HPLC peaks (1 and 2). Fractions were analyzed by SDS-PAGE on a 14% gel
and silver-stained. The molecular masses of standard proteins in kDa
are indicated to the left.
T28 and E31 are overlapping fragments of eIFiso4G
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Fig. 4.
Peptide start sites and amino acid sequence
comparison of eIF4G isoforms from various species. A,
localization of the start sites for the phosphocellulose
(T28) and oligo(dT)-cellulose (E31) binding
domains of wheat eIFiso4G (wheat i4G), and for the
homologous domain from wheat eIF4G (4Gpep,
wheat 4G). The eIFiso4G sequence is shown from
Gln170 through Leu218, with the corresponding
eIF4G sequence below (see Footnote 2). Residues obtained from
N-terminal sequencing are double underlined. The
second block of homology shared by mammalian and plant factors begins
at Lys215 in eIFiso4G (Region 2). B,
conserved sequences around the start site of the oligo(dT) binding
domain are shown in bold. Cress, thale cress
(Arabidopsis thaliana); i4G, eIFiso4G;
4G, eIF4G; 4G2, eIF4G2. Where known, amino acid
residues are numbered at the right. A consensus
sequence is given, with spacing between the conserved residues
calculated as the average number of intervening amino acids from all
species except yeast.
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Fig. 5.
Competitive elution of eIFiso4F from
oligo(dT)-cellulose by nucleic acids. eIFiso4F was applied to
oligo(dT)-cellulose spin columns, which were successively washed with
B50, either 200 µM oligo(dT)12
(top) or oligo(dT)6 (bottom), and
with B200. i4F, eIFiso4F; i4G, eIFiso4G;
i4E, eIFiso4E; FT, flow-through fraction from
protein application.
Nucleic acid binding specificity of eIFiso4F and the E31 domain of
eIFiso4G
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Fig. 6.
Specific binding of E31 to
poly(G)-agarose. Purified E31 was applied to a poly(G)
spin column and washed successively with B50, 1 mM GTP,
B100, and B200. Only selected fractions are shown. FT,
flow-through from E31 application.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Comparison of binding, activity, and
structural domains in eIFiso4G and eIF4G. Schematics of wheat
eIFiso4G (wheat i4G) and human eIF4G (human 4G)
are shown. The six regions of homology identified previously (6) are
represented by black boxes in the background. The homologous
sequence at the N terminus of the protease-resistant central domain,
from Lys192 to Arg209 of wheat eIFiso4G, is
backed by an open box. Wheat i4G, sites for
binding eIFiso4E and eIF4A (i4E/4A) and for ATPase activity
(ATPase) (12) are indicated by white boxes within
the schematic. Human 4G, the binding site for eIF4E
(4E) (9) is indicated by a white box within the
schematic, and regions which bind eIF3 (10) and eIF4A (10, 11) are
labeled. The locations of T28 and E31 within
wheat i4G are represented by black lines above
the factor schematic, and their binding specificities for
phosphocellulose (PC) and nucleic acids (NA) are
indicated.
The central third of eIF4G is known to have several important functions. First, Pestova et al. (15) have shown that eIF4A and the central third of eIF4G are alone sufficient for cap-independent initiation of encephalomyocarditis virus RNA, and that the eIF4G fragment cross-links to the viral IRES. The wheat eIF4G and eIFiso4G core domains identified here would be included in this larger 476-amino acid fragment, and could be involved in IRES recognition or binding.
Second, it has been known for some time that chromatography of cap-binding proteins on phosphocellulose depletes the complex of eIF4A. The extent to which this occurs varies between laboratories. We have found that, for the plant factors, eIF4F is substantially depleted of eIF4A, while eIFiso4F is completely stripped of eIF4A, both on phosphocellulose (Fig. 1) and oligo(dT)-cellulose (data not shown). Why the binding of eIFiso4G to eIF4A and phosphocellulose should be mutually exclusive was not clear, given the lack of overlap in their recognition sites (Fig. 7); the eIF4A binding site on wheat eIFiso4G was mapped to the N terminus of the molecule by Metz and Browning (12), using eIFiso4G deletion constructs expressed in a yeast two-hybrid system. In contrast, two eIF4A binding domains have now been identified in mammalian eIF4G, one in the C-terminal third of the molecule (10), and a second in the central third (11). Only the eIF4A binding site in the central third of eIF4G is conserved in eIFiso4G, and mutations in this sequence of the mammalian factor abolished eIF4A binding activity (11). This site corresponds to the second block of homology shared by eIF4G and eIFiso4G (Fig. 4, Region 2; Fig. 7). As RNA recognition motifs are typically 50-100 amino acids long (31), the eIF4A and nucleic acid binding sites may well overlap, with the loss of eIF4A during chromatography resulting from direct competition. This has interesting implications for the mode of action of the mRNA cap-binding complexes, which perhaps deliver eIF4A to the message, and then bind mRNA directly through the same site to stabilize the preinitiation complex.
Third, Lamphear et al. (10) have shown that a ~407-amino acid peptide from the central third of eIF4G binds to eIF3 (Fig. 7). Further proteolysis, resulting in the removal of ~50 amino acids from the C terminus of the peptide, abolished this activity. Thus, at least part of the eIF3 recognition sequence in the mammalian factor lies carboxyl to and outside of the core domain defined here, while the nucleic acid binding site in both plant factors begins at the N-terminal boundary of this structural core. Based on these observations, it seems unlikely that these two sites overlap.
Finally, one can speculate on why a translation factor involved in the
binding and recruitment of mRNA to ribosomes should also bind ss-
and dsDNA. Such dual specificity in RNA-binding proteins is not
unusual, and may have no biological significance. However, several
examples of RNA-binding proteins that recognize specific DNA sequences
have now come to light (32-34). The cap-binding translation factors
may also have multiple functions. eIF4E has been found localized to the
nucleus in both mammalian (35) and yeast cells (36), and it appears to
regulate the expression of cyclin D1 at both the transcriptional and
post-transcriptional levels (37). eIF4E has also been shown to
cooperate with two oncogenes, v-myc and E1A, in
the transformation of primary fibroblast cultures (38). Additional
functions for eIFiso4G have also been reported. eIFiso4G was shown to
bind, bundle and co-localize with microtubules in maize cells (18), and
to promote their growth by end-to-end annealing in vitro
(24), suggesting possible roles for eIFiso4F in the localization of
translational components to the cytoskeleton (18) or in mRNA
transport (1). These relatively ancient RNA-binding proteins may
therefore have other functions in both the nucleus and cytoplasm,
beyond formation of the 43 S preinitiation complex in translation.
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ACKNOWLEDGEMENTS |
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We thank Karen S. Browning for sharing unpublished sequence information for wheat eIF4G; Rod Nakayama for the preparation of figures; and Fred Kim, Greg Kim, Rick Kim, Dimitri Niks, David Park, Kenny Park, Tung Nguyen, and John Wingo for factor preparation and technical assistance.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB-9305713 (to C. W.) and United States Department of Agriculture NRI-CGP Grant 98-35100-6146 (to J. K. M. R.).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.
Current address: Los Alamos National Laboratory, Life Science-6,
Los Alamos, NM 87545.
§ Current address: Mitsui Plant Biotechnology Research Institute, Sengen 2-1-6, Tsukuba, Ibaraki 305-0047, Japan.
¶ Current address: College of Pharmacy and Pharmacal Sciences, Howard University, Washington, DC 20059.
To whom correspondence should be addressed. Tel.:
909-787-2412; Fax: 909-787-3590; E-mail: websterc{at}mail.ucr.edu.
2 S. Manjunath, C. Webster, and J. Bailey-Serres, unpublished sequence information.
3 S. D. Rounsley, X. Lin, S. Kaul, T. P. Shea, C. Y. Fujii, T. M. Mason, M. Shen, C. M. Ronning, C. M. Fraser, C. R. Somerville, and J. C. Venter (1998) GenBank direct submission.
4 Unpublished sequence information for wheat eIF4G was a personal communication (M. L. Allen and K. S. Browning).
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ABBREVIATIONS |
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The abbreviations used are: eIF, eukaryotic initiation factor; m7GTP, 7-methyl-GTP; RP, reverse phase; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; aa, amino acid(s); ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; IRES, internal ribosome entry site. The nomenclature for mammalian and yeast translation factors was recently revised by a panel appointed by the IUBMB Nomenclature Committee, Marianne Grunberg-Manago, Convener. In keeping with these guidelines, parentheses and hyphens have been deleted, and the large and small subunits of eIFiso4F, the plant-specific isoform of eIF4F, are designated herein as eIFiso4G and eIFiso4E, rather than p86 and p28.
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REFERENCES |
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