(Received for publication, August 1, 1995; and in revised form, September 21, 1995)
From the
The binding of p28, p86, and native wheat germ eIF-(iso)4F with
mGTP and oligonucleotides was measured and compared. The
purified subunits (p28, 28 kDa and p86, 86 kDa) of wheat germ protein
synthesis initiation factor eIF-(iso)4F have been obtained from Escherichia coli expression of the cloned DNA (van Heerden,
A., and Browning, K. S.(1994) J. Biol. Chem. 269,
17454-17457). The binding of the 5`-terminal cap analogue
m
GTP to the small subunit (p28) of eIF-(iso)4F as a
function of pH, temperature, and ionic strength is described. The mode
of binding of p28 to cap analogues is very similar to the intact
protein. Assuming that all tryptophan residues contribute to p28 and
eIF-(iso)4F fluorescence, iodide quenching shows that all 9 tryptophan
residues in p28 are solvent-accessible, while only 6 out of 16
tryptophan residues are solvent-accessible on the intact eIF-(iso)4F.
The fluorescence stopped-flow studies of eIF-(iso)4F and p28 with cap
show a concentration-independent conformational change. The rate of
this conformational change was approximately 10-fold faster for the
isolated p28 compared with the native eIF-(iso)4F. From these studies
it appears that cap recognition resides in the p28 subunit. However,
p86 enhances the interaction with capped oligonucleotides and probably
is involved in protein-protein interactions as well. Both subunits are
required for helicase activity.
Recognition of the m-cap structure of mRNA by
eucaryotic initiation factors and formation of 48 S initiation complex
are important steps in initiation of protein synthesis in eucaryotic
cells. In mammalian cells, formation of the 48 S initiation complex is
catalyzed by initiation factors eIF(
)-4A, eIF-4B, eIF-4E,
and eIF-4
. The complex termed eIF-4F contains polypeptides eIF-4A,
eIF-4E, and eIF-4
(Grifo et al., 1983; Edery et
al., 1983), but a complex of only eIF-4E and eIF-4
has also
been determined (Etchison and Milburn, 1987; Buckley and Ehrenfeld,
1987). In addition, eIF-4E can be isolated as a separate polypeptide
(Rinker et al., 1992). Recent evidence suggests that eIF-4E
and eIF-4
form a complex concurrent with the binding of mRNA to
the 40 S ribosomal subunit (Joshi et al., 1994). Such a
stepwise addition suggests specific functional roles for the separated
polypeptides. In contrast to the mammalian eIF-4F complex, the
individual polypeptides of the eIF-4F complex from wheat germ cannot be
separated except under denaturing conditions. Wheat germ eIF-(iso)4F is
one of the two wheat germ initiation factors that has cap binding
ability, the other is wheat germ eIF-4F. Wheat germ eIF-(iso)4F
consists of two subunits (p28 and p86) in a 1:1 molar ratio (Lax et
al., 1985), while wheat germ eIF-4F consists of a 26-kDa and a
220-kDa subunit in a 1:1 molar ratio. Some structural and functional
similarity exists between these two factors. Both wheat germ eIF-4F and
eIF-(iso)4F have RNA-dependent ATPase activity. Only one wheat germ
factor is required for ATP hydrolysis and stimulation of protein
synthesis in an eIF-4F or eIF-(iso)4F deficient translation system (Lax et al., 1985, 1986b). Wheat germ eIF-(iso)4F can substitute
for mammalian eIF-4F in an RNA-dependent ATPase activity and in
cross-linking of mammalian eIF-4A to the cap of oxidized mRNA (Abramson et al., 1988).
We have obtained purified subunits of wheat germ eIF-(iso)4F from expression in E. coli and examined the binding and functional properties of the separated subunits. Separate subunits of eIF-(iso)4F were not functionally active as measured by the ability to stimulate polypeptide synthesis. However, when both subunits were added, activity was equal to native eIF-(iso)4F (van Heerden et al., 1994). These results indicate that the two subunits are able to associate to form an active complex. In addition, both subunits are required for activity. In order to understand the RNA binding ability of the intact protein and the function of the individual subunits, we have studied the binding and physical properties of the separated subunits, reconstituted protein, and native eIF-(iso)4F.
Buffer A, used for fluorescence measurements, consisted of 20
mM HEPES-KOH, 100 mM KCl, 1 mM dithiothreitol, and 1 mM MgCl and was
adjusted to the appropriate pH. Buffer B, used in isolation of the
wheat germ factors, consisted of 20 mM HEPES-KOH, pH 7.6, 0.1
mM EDTA, 1 mM dithiothreitol, 10% glycerol, and KCl
as indicated. All chemicals were reagent grade or better.
m
GTP and m
GpppG were purchased from Sigma (St.
Louis, MO). m
GTP-Sepharose was obtained from Pharmacia
Biotech Inc. mRNA analogues (capped and uncapped oligonucleotides) were
transcribed from partially double-stranded deoxyoligonucleotide
templates containing a T7 RNA polymerase primer according to the
procedures of Milligan et al.(1987) and were purified
according to the method of Draper et al.(1988); the
oligonucleotides used for the binding study, I and II, are shown in Fig. 1. The oligonucleotides used for the helicase assay, III
and IV, are shown below.
Figure 1: Structures of oligonucleotides I and II.
Wheat germ initiation factor eIF-(iso)4F
was purified according to the protocol of Lax et al. (1986a,
1986b) with the following modifications. The 40% ammonium sulfate
fraction was loaded onto a DE52 column, equilibrated with buffer B
containing 40 mM KCl (B-40). The column was washed until the
OD was less than 0.2. The buffer was then changed to B-80 (80 mM KCl), and the peak containing eIF-(iso)4F was collected and
concentrated with 80% ammonium sulfate precipitation and centrifuged at
11,000 rpm in a Sorvall centrifuge; the precipitate, which contains
eIF-(iso)4F, was resuspended in 10 ml of B-120 and dialyzed overnight
against B-120. The sample was then applied to an
mGTP-Sepharose column, and the pure factor was eluted as
described by Lax et al. (1986a, 1986b); in order to maximize
yields, the m
GTP solution used to elute the pure
eIF-(iso)4F factor was freshly prepared. The pure p28 and p86 subunits
were prepared as described previously (van Heerden et al.,
1994).
Fluorescence measurements were carried out at 23 °C, unless otherwise noted, and data were collected and analyzed as described previously in detail (Carberry et al., 1989, 1990). Background fluorescence emission was subtracted.
The double-stranded
RNA (T = 65 °C) for the helicase assay
of eIF-(iso)4F was prepared by annealing the partially complementary
III and IV single-stranded RNA. Equal molar amounts of RNA III and IV
were mixed and heated to 80 °C for 10 min, slowly cooled to 50
°C for 30 min, and was then cooled to 40 °C and kept overnight.
The double-stranded RNA is shown as
follows.
On-line formulae not verified for accuracy
The unwinding reaction for the helicase assay was performed by
incubating partially double-stranded RNA with eucaryotic initiation
factors (5 µM eIF-4A, 2 µM p28, p86, and
eIF-(iso)4F were used) in 20 mM Tris-HCl buffer (pH 7.5)
containing 2 mM ATP, 1 mM magnesium acetate. The
reaction mixture was incubated at 37 °C for 20 min and then
transferred to an ice bath and 2 ml of 5 stopping solution
containing 50% glycerol and 10% SDS was added. SDS was used to separate
the protein from the RNA and to avoid the formation of a large
molecular weight complex. The sample was loaded onto a 15%
nondenaturing polyacrylamide gel. The double- and single-stranded RNA
on the gel was silver-stained (Bassam et al., 1991), and the
bands were quantitatively determined by a laser scanning densitometer.
Stopped-flow fluorescence experiments were performed on a Hi-Tech
SF-51 stopped-flow spectrofluorometer equipped with a Berger-type
mixing chamber and a 2 2
10-mm flow cell with a dead
time
1.5 ms. The excitation wavelength was 280 nm, and the slit
width was 2 mm. Light emitted from the reaction mixture was monitored
after passage through a cut-off emission filter (WG-320 provided by
Hi-Tech). A series of stopped-flow experiments were carried out at 23
°C in buffer A, pH 7.05, under different concentrations of cap
analogue m
GpppG. After rapid mixing of the protein with the
mRNA cap, the time course of the intrinsic fluorescence intensity was
recorded.
Figure 2:
Fluorescence emission spectra of p28
subunit of wheat germ eIF-(iso)4F (0.5 µM) titrated with
capped oligonucleotide II in buffer A (pH 7.05) at 23 °C. The
oligonucleotide concentration (top to bottom) was 0,
0.1, 0.3, 0.6,1.2 and 2.3 µM. The excitation wavelength
was 282 nm, and a 1.4-mm slit was employed. Emission maxima was
observed at 331 nm. Inset, Eadie-Hofstee plot of fluorescence
data. F was calculated at 331 nm, where
F = F
- F
+ oligonucleotide
The K of
p28
oligonucleotide and p28
m
GTP complex
formation can be calculated from the relative fluorescence intensity
changes in free and complexed p28 emission spectra by construction of
an Eadie-Hofstee plot, as shown in the inset of Fig. 2. K
was found to be (1.24 ± 0.04)
10
M
for oligonucleotide II in Fig. 2.
Figure 3:
Binding of mGTP and p28 as a
function of pH. All solutions were prepared in buffer A, adjusted to
the appropriate pH at 23 °C. Other conditions were the same as
described in the legend to Fig. 2.
Figure 4:
Van't Hoff plot for
mGTP
p28 interactions. All experimental conditions
were the same as described in the legend to Fig. 2.
Figure 5:
Debye-Huckel analysis of Keq data
(, KCl dependence;
,
KC
H
O
dependence).
Figure 6:
Modified Stern-Volmer plot of iodide
quenching to p28 () and to eIF-(iso)4F
(
).
On-line formulae not verified for accuracy
Figure 7:
Single exponential curve fitting of the
F versus time for the kinetics of 0.5 µM eIF-(iso)4F protein mixing with 5 µM m
GpppG cap analogue. k
is
obtained from the fitted curve as 11.4 s
. The bottom inset shows the residuals of fitting amplified by
4-fold and plotted on the same
and y axis.
where k is the observed first-order rate
constant, and the
F
is the maximum
fluorescence change.
The one-step reaction is as follows,
On-line formulae not verified for accuracy
where k and k
are forward and reverse rate constants, respectively; P and C
refer to eIF-(iso)4F and m
GpppG, respectively. Under the
pseudo first-order condition, the observed rate constant is predicted
to be a linear function of substrate concentration, i.e.
k
= k
[C]
+ k
.
The two-step reaction is as follows,
On-line formulae not verified for accuracy
which involves a fast association of protein, eIF-(iso)4F, and
cap analogue mGpppG followed by a slow change of
conformation of the first association complex, (P
C)*, to the
stable complex, P
C, giving rise to the fluorescence change.
The interaction of eIF-(iso)4F (0.5 µM) with
mGpppG under different concentrations of cap analogue (2.5,
5, and 10 µM) gave about the same k
(10.7, 11.5, and 12.1 s
, respectively). The
reaction is not a single-step pseudo first-order reaction (Mechanism
i). Earlier CD experiments have shown that eIF-(iso)4F undergoes
conformational changes upon binding to m
GpppG (
)in agreement with Mechanism ii. Mechanism ii can be
written as follows (Olsen et al.,
1993),
On-line formulae not verified for accuracy
where K = k
/k
. rearranges as
follows.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
A plot of 1/kversus 1/[C] will give an intercept of 1/k
(Fig. 8).
Figure 8:
Kinetics plot of 1/kversus 1/[C] of 0.5 µM eIF-(iso)4F (
) and p28 (
) under different
m
GpppG concentrations (2.5, 3.3, 5, and 10 µM,
respectively). k
was obtained as the reciprocal of
the y intercept (12.2 ± 0.5 s
for
eIF-(iso)4F and 123.3 ± 8.6 s
for
p28).
This model gives a k value of 12.2 ± 0.5 s
.
The general mechanism of interaction of the cap with the p28
subunit of eIF-(iso)4F is very similar to the wild type protein. The pH
optimum for cap binding to p28 was found to be 7.05; compared with the
pH optimum of 7.6 for eIF-(iso)4Fm
GpppG complex
(Carberry et al., 1991). The pK
values of
amino acids are known to be environmentally sensitive and this could
account for the shift in binding profile between p28 and eIF-(iso)4F.
The shift in pK
implies the binding site has
become more positively charged in the separated subunit. The previous
sequence comparison (Allen et al., 1992) of wheat p28 with the
cap-binding protein eIF-4E from mammals and yeast had shown about 38%
sequence homology. There are three highly conserved histidine residues
(position 33, 91, 193 from NH
-terminal) and 9 tryptophan
residues in p28 (positions from NH
-terminal: 39, 42, 55,
72, 101, 112, 126, 161, 178), one more tryptophan residue than in
mammalian and yeast cap-binding proteins (Trp-178). Site-directed
mutagenesis of yeast eIF-4E showed that tryptophan 1, 2, and 8 were
required for cap binding activity (Altmann et al., 1988).
Site-directed mutagenesis of human eIF-4E showed that tryptophan 5 and
the glutamic acid residue three amino acids to the carboxyl-terminal
side of tryptophan 5 were involved in cap recognition. None of these
studies examined the tertiary structure of the mutant protein; however,
Ueda et al.(1991) proposed that the base stacking of the
tryptophan and hydrogen-bond pairing by the glutamic acid are
responsible for binding the m
G cap of mRNA. The p28 of
eIF-(iso)4F has a glutamic acid residue four amino acids to the
carboxyl-terminal side of tryptophan 5 (Glu-105) which could
participate in the binding proposed by Ueda et al.(1991).
Their data, however, fail to account for the pH dependence of binding.
The small subunit, p28, has cap binding properties very similar to
native eIF-(iso)4F. This observation is similar to the relative
affinities of mammalian eIF-4E and eIF-4F binding to globin mRNA
(eIF-4E: K = (20.9 ± 1.0)
10
M
; eIF-4F: K
= (18.6 ± 1.1)
10
M
, Goss et al., 1990). In the
mammalian system eIF-4E has about the same affinity for mRNA as the
larger eIF-4F complex. These data suggest that the essential role of
the large subunit is involvement in other interactions such as
protein-protein interactions and helicase activity.
The p86 subunit,
which also binds to mGTP, although less tightly, is
unlikely to be the specific cap binding subunit in eIF-(iso)4F for the
following two reasons: p86 shows no significant difference between
capped RNA, and uncapped RNA binding and p86 does not distinguish
m
GTP from GTP. The fact that p86 has a relatively high
binding affinity for RNA suggests the native protein may have an RNA
site involving both subunits and that p86 stabilizes the contact with
RNA during the course of protein synthesis. In addition, photoaffinity
labeling with m
GTP analogues label only the small subunit. (
)
The stopped-flow kinetic data showed that the small
subunit, p28, changed conformation approximately 10 times faster than
eIF-(iso)4F (p28: k = 123.5 ± 8.6
s
; eIF-(iso)4F: k
= 12.2
± 0.5 s
). The faster conformational change
rate of the p28-cap may be partially caused by an agile movement of its
small mass (p28, 28 kDa compared with eIF-(iso)4F, 110 kDa). These
results are surprising, since one would expect p86 to increase the rate
of complex formation and hence increase the rate of interaction.
However, if the initial equilibrium between the cap and protein is
approximately the same for p28 and eIF-(iso)4F, then the off rate for
eIF-(iso)4F would also be much slower than the off rate for p28. This
would allow for interaction of other initiation factors or ribosomes
and favor formation of an active initiation complex.
A working model is that p28 contains the binding site for the cap and locates the complex at or near the 5` end of the mRNA. While p86 does not affect the equilibrium affinity for cap, it does affect the rate of complex formation. In order for the eIF-(iso)4F complex to perform helicase activity (which requires both p28 and p86), the complex must process down the RNA. This would in all probability require release of the cap while maintaining contact with the RNA. p86 and other factors may stabilize the interaction with mRNA and prevent the protein complex from dissociating from the RNA. While p28 binds capped RNA specifically, the intact protein (or sum of p28 + p86) is necessary to stimulate protein synthesis (van Heerden et al., 1994). This suggests p86 is involved in protein-protein interactions to form a complete initiation complex.