Functional reconstitution of human eukaryotic
translation initiation factor 3 (eIF3)
Chaomin Suna,1, Aleksandar Todorovica,1, Jordi Querol-Audía, Yun Baib, Nancy Villac, Monica Snyderd, John Ashchyanb,
Christopher S. Lewise, Abbey Hartlanda, Scott Gradiaa, Christopher S. Fraserc, Jennifer A. Doudnab,f,g,2, Eva Nogalesb,f,h,
and Jamie H. D. Cateb,d,g,2
aCalifornia Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;
California, Berkeley, CA 94720;
University of California, Berkeley, CA 94720;
fHoward Hughes Medical Institute, University of California, Berkeley, CA 94720;
Berkeley, CA 94720; and
bDepartment of Molecular and Cell Biology, University of
dDepartment of Chemistry,
cDepartment of Molecular and Cellular Biology, University of California, Davis, CA 95616;
eDepartment of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720;
gPhysical Biosciences Division, Lawrence Berkeley National Laboratory,
hLife Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
Contributed by Jennifer A. Doudna, October 12, 2011 (sent for review September 25, 2011)
Protein fate in higher eukaryotes is controlled by three complexes
that share conserved architectural elements: the proteasome, COP9
signalosome, and eukaryotic translation initiation factor 3 (eIF3).
Here we reconstitute the 13-subunit human eIF3 in Escherichia coli,
revealing its structuralcoreto be the eight subunitswith conserved
orthologues in the proteasome lid complex and COP9 signalosome.
This structural core in eIF3 binds to the small (40S) ribosomal sub-
unit, to translation initiation factors involved in mRNA cap-depen-
dent initiation, and to the hepatitis C viral (HCV) internal ribosome
entry site (IRES) RNA. Addition of the remaining eIF3 subunits
enables reconstituted eIF3 to assemble intact initiation complexes
with the HCV IRES. Negative-stain EM reconstructions of reconsti-
tuted eIF3furtherrevealhowthe approximately400 kDamolecular
mass structural core organizes the highly flexible 800 kDa molecu-
lar mass eIF3 complex, and mediates translation initiation.
protein synthesis ∣ translation regulation ∣ supramolecular complex
assembly ∣ electron microscopy
teins in higher eukaryotes. These complexes, the proteasome,
COP9 signalosome, and eukaryotic translation initiation factor
3 (eIF3), each contain eight conserved protein subunits, six of
which bear proteasome, COP9, eIF3 (PCI) domains and two of
which bear Mpr1-Pad1 N-terminal (MPN) domains (1), here
grouped as PCI/MPN subunits. Although interaction maps have
been proposed for each complex (2–5), their structures, assembly
pathways, and the functional contributions of subunits within
them remain poorly understood (1, 5–7). A major barrier to the
study of these macromolecular protein regulators has been the
inability to produce samples in genetically tractable recombinant
systems in sufficient quantity for mechanistic studies.
Among these three complexes, eIF3 remains the least charac-
terized, despite its central role in recruiting both mRNAs and
the cellular translation machinery to form translation initiation
complexes (8). In humans, eIF3 is an 800 kDa molecular mass
assembly of 13 proteins (eIF3a-eIF3m) (3, 9), many of which have
been implicated in cancers via misregulation of their expression
(10). In addition to its involvement in regulation of cell prolifera-
tion, eIF3 also interacts directly with the hepatitis C viral (HCV)
mRNA to promote the translation of viral proteins (11–13). The
5′-untranslated region (5′-UTR) of HCV contains an approxi-
mately 340-nt internal ribosome entry site (IRES) element that
folds into defined secondary and tertiary structural elements (14)
capable of specifically binding to eIF3 and the 40S ribosomal
subunit (13, 15). These interactions trigger HCV IRES-mediated
initiation complex formation with the GTPase eIF2 and methio-
nyl-initiator tRNA (Met-tRNAi) (16, 17) or an alternate initia-
tion complex with eIF5B (18).
hree protein complexes with conserved composition and
architectures regulate the synthesis and degradation of pro-
A structural and functional understanding of eIF3’s roles in
human translation initiation has relied on human eIF3 purified
from human cell lines (19–21), expressed in part in insect cells
(3, 21, 22), or by comparison to eIF3 purified from other mam-
malian sources (18, 23). Human eIF3 purified from human cell
lines was used to derive a low-resolution structural model of
the complex and its interactions with the small (40S) ribosomal
subunit and HCV IRES (24). Attempts to reconstitute human
eIF3 expressed in insect cells revealed parallels between the hu-
man and yeast eIF3 complexes, which share six conserved subu-
nits (21). Additional reconstitution experiments in insect cells led
to the model that the functional core of human eIF3 is composed
of six subunits (eIF3a, eIF3b, eIF3c, eIF3e, eIF3f, and eIF3h)
of which only eIF3a, eIF3b, and eIF3c are conserved in all eukar-
yotes (22). However, a full molecular understanding of eIF3’s
roles in translation remains to be determined.
Here we have reconstituted human eIF3 in a stepwise manner
using expression in Escherichia coli, resulting in a 13-subunit
recombinant form of eIF3. We have tested the resulting sub-
complexes and the 13-subunit recombinant eIF3 for their ability
to bind components of the translational machinery, to the HCV
IRES RNA, and to form initiation complexes. Finally, we com-
pare the structures of the eIF3 complexes assembled using E. coli
expression with that determined for natively purified human
eIF3. Our results establish a unique paradigm for expression of
intact human supramolecular assemblies in E. coli, and reveal
the molecular basis for eIF3 assembly, structure, and function
in translation initiation. This analysis also reveals architectural
features likely to be shared between the proteasome, COP9
signalosome, and eIF3 and suggests a productive strategy for me-
chanistic and structural dissection of these and other large macro-
molecular assemblies that control the eukaryotic proteome.
Stepwise Reconstitution of Human eIF3. We tested whether sub-
complexes identified during eIF3 disassembly (3), or assembled
using baculovirus expression (21, 22) could be formed de novo
Author contributions: C.S., A.T., J.Q.-A., Y.B., J.A.D., E.N., and J.H.D.C. designed research;
C.S., A.T., J.Q.-A., Y.B., M.S., J.A., and C.S.L. performed research; N.V., A.H., S.G., and C.S.F.
contributed new reagents/analytic tools; C.S., A.T., J.Q.-A., Y.B., N.V., M.S., J.A., C.S.L., A.H.,
S.G., C.S.F., J.A.D., E.N., and J.H.D.C. analyzed data; and C.S., A.T., J.Q.-A., A.H., S.G., J.A.D.,
and J.H.D.C. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The negative-stain EM maps of the eIF3 PCI/MPN octamer and eIF3
dodecameric complex have been deposited in the Electron Microscopy Data Bank,
www.ebi.ac.uk/pdbe/emdb/, (EMDB accession codes EMD-1975 and EMD-1976).
1C.S. and A.T. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: email@example.com or jcate@
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1116821108PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20473–20478
in E. coli. Subunits eIF3a and eIF3c truncated to remove likely
flexible regions of the proteins (Fig. 1A) (25) yielded a stable a*c*
dimer (asterisks indicating the truncations, Fig. 1A) that further
assembled with subunits containing PCI (subunits a, c, e, k, l,
and m) and MPN (subunits f and h) domains (Fig. S1), as well as
with subunits conserved in all eukaryotes (b, g, and i) (21, 26–28)
(Fig. S1). These results indicate that the a*c* dimer is central
to eIF3, as it interacts with each of the remaining PCI and
MPN domain-containing subunits in human eIF3, and also as-
sembles with the universally conserved bgi subcomplex. However,
the resulting trimeric through heptameric complexes were not
biochemically stable, and were highly prone to aggregation, as
determined by gel filtration chromatography.
Coexpression of the eight subunits that contain PCI or MPN
domains (1) resulted in a highly stable octameric a*c*efhklm, or
PCI/MPN, complex (Fig. 1 B and C) that further assembled into
larger recombinant human eIF3 complexes in vitro. Subunits d
and b purified individually, and subunits g and i coexpressed with
each other (3, 22, 26, 27), added serially to the PCI/MPN octamer
formed 9–12 subunit complexes, respectively (Fig. 1 B and C).
The final subunit eIF3j associates with the remainder of eIF3
in a salt-dependent manner (21), and bound well to recombinant
eIF3c*, the a*c* dimer, and the PCI/MPN octameric complex
(Fig. 1D), as well as to subunits f and h (Fig. S1). Addition of
eIF3j to the recombinant dodecameric eIF3 formed a complete
13-subunit human eIF3 of approximately 700 kDa molecular
mass, lacking only the truncated domains in subunits eIF3a
and eIF3c (Fig. 1 A and D).
Function of Reconstituted eIF3 Assemblies Expressed in E. coli. To test
the function of reconstituted eIF3 complexes, we assessed each
complex for its ability to bind components of the translation in-
itiation machinery and the HCV IRES. The biochemically stable
PCI/MPN complex bound tightly to the IIIabc RNA (apparent Kd
of 20–40 nM) (Fig. 2A), similar to natively purified eIF3 and to
the 13-subunit reconstituted eIF3 (Fig. S2). An a*c*efhl hexamer
(Fig. S1B) also bound tightly to the IIIabc RNA (Fig. S2) (25),
but was biochemically unstable and highly prone to aggregation.
Addition of subunits k or m individually to the hexamer did not
affect IIIabc domain binding (Fig. S2) or improve the biochem-
ical stability of the subcomplexes.
The PCI/MPN octamer also bound tightly to the 40S ribosomal
subunit, with an apparent affinity two- to threefold weaker
than that of natively purified human eIF3 (Fig. 2B, Fig. S3). The
addition of subunits d, b, and the gi dimer to the octamer did not
increase the apparent binding affinity of the recombinant eIF3
complexes for the 40S subunit (Fig. S3). Including eIF3j, which
stabilizes eIF3 binding to the 40S subunit on sucrose gradients
(21, 29), had little impact on the apparent affinity of recombinant
and natively purified eIF3 complexes for the 40S subunit in native
gels (Fig. S3). Initiation factors eIF1 and eIF1A, which bind
to eIF3 (28, 30, 31) and aid the formation of stable preinitiation
complexes with the 40S subunit (32), also bind to the PCI/MPN
core in affinity pull-down assays (Fig. 2C).
Translation initiation factor eIF4G in humans binds tightly
to eIF3 (33, 34), and is positioned near the mRNA exit site of the
40S subunit (24). Although the central domain of eIF4G did not
bind to the PCI/MPN octamer (Fig. S4), it did bind to a 10-sub-
unit complex that includes the octamer plus subunits b and d
(Fig. S4). Notably, binding of eIF4G to the 10-subunit complex
in some experiments resulted in loss of eIF3b during the affinity
pull-down (Fig. 2D), due to eIF3b dissociation from the complex
under the conditions used. Thus eIF4G likely interacts with the
9-subunit eIF3 complex containing the PCI/MPN core and sub-
unit d, after a conformational change induced in the complex by
To test whether reconstituted eIF3 forms intact HCV IRES-
mediated initiation complexes, we programmed rabbit reticulo-
cyte lysate (RRL) with an mRNA containing the HCV IRES.
Affinity purification of reconstituted and GST-tagged eIF3 from
RRL reactions that included 2 mM GMPPNP (35) showed that
reconstituted 12-subunit eIF3 could form preinitiation complexes
with the 40S subunit and eIF2∕Met-tRNAi(Fig. S5) as well as
HCV IRES-mediated initiation complexes (Fig. 3, Fig. S6) (15).
The 9-subunit eIF3 containing the PCI/MPN octamer and GST-
eIF3d was less efficient at forming HCV IRES-mediated initia-
tion complexes (Fig. S7), indicating that eIF3 subunits b, g, and
i are important for HCV IRES-driven translation initiation.
Structuresof thePCI/MPN OctamerandReconstituted eIF3Complexes.
Given the significant number of activities associated with the
eIF3 PCI/MPN octamer, we wondered how its structure com-
pares to that of natively purified eIF3. In an approximately 23 Å
negative-stain reconstruction, the PCI/MPN octamer bears a
striking resemblance to the previously determined cryoEM struc-
ture of native human eIF3 (24), as well as to the reconstituted
12-subunit eIF3 at a resolution of approximately 29 Å (Fig. 4A).
This result is a surprise, as the PCI/MPN octamer is just over
400 kDa in molecular mass, compared to the 800 kDa molecular
mass of native human eIF3 used in prior EM reconstructions
(24), and the 700 kDa molecular mass of the reconstituted
12-mer. Quantitative difference maps between the reconstruc-
tions of the PCI/MPN octamer and reconstituted 12-subunit
eIF3 show two major regions of additional density in the larger
complex, at the end of the left arm and leg features (Fig. 4B). This
difference density cannot account for the approximately 300 kDa
in molecular mass missing in the PCI/MPN octamer, suggesting
eIF3a and eIF3c, denoted a* and c*. All other subunits were full-length (9). (B)
Purification scheme for 12-subunit eIF3, beginning with the purified PCI/MPN
octamer. Purifications of untagged 9-mer and 10-mer complexes similarly
used TEV cleavage of the GST tag and gel filtration, when needed. Details
are in SI Materials and Methods. (C) Coomassie blue-stained SDS gel of
the octameric PCI/MPN complex containing subunits a*c*efhklm, eIF3 nona-
mer (a*c*defhklm), eIF3 decamer (a*bc*defhklm), and eIF3 dodecamer
(a*bc*defghiklm), affinity-purified sequentially starting from the PCI/MPN
octameric core using GST-tagged eIF3d, and with added subunits (bold) ex-
pressed in E. coli. Molecular weight (MW) markers, in kilodaltons, are shown
to the left, and subunit positions are marked to the right. (D) Native agarose
gel of eIF3 complexes showing binding of eIF3j. Fluorescently labeled eIF3j
(10 nM) was incubated with the other eIF3 subunits or complexes (30 nM)
prior to loading the gel. In the absence of binding, eIF3j does not enter
the gel as a discrete band (lane 1). Lane 1, eIF3j alone; lanes 2–6, eIF3j incu-
bated with other recombinant eIF3 components (lanes 2–5), or native human
eIF3 depleted of eIF3j (lane 6).
Subassemblies of eIF3 expressed in E. coli. (A) Truncations in subunits
www.pnas.org/cgi/doi/10.1073/pnas.1116821108Sun et al.
that a significant part of the added subunits (b, d, g, and i) exist in
multiple conformations that average out during the reconstruc-
Our approach of combining gene synthesis, multisubunit coex-
pression in E. coli, and stepwise assembly provides a framework
for dissecting human eIF3 structure and function. In the assembly
pathway for human eIF3 reconstituted here, a dimer of subunits
eIF3a and eIF3c serves as a central scaffold to which most of the
other subunits bind. Notably, the a*c* dimer directly interacts
with all of the remaining PCI/MPN domain-containing subunits
individually, as well as with eIF3b and eIF3j (Fig. S1). Although
some of these interactions had been inferred from prior genetic,
biochemical, and disassembly experiments (3, 36), the present
results provide direct evidence for many of these interactions
(Fig. 4C). Additionally, the core of eIF3 is only stable when all
domain, schematically drawn to the right. The nanomolar concentrations of the octamer are listed. Lane 1 shows IIIabc RNA binding to natively purified human
eIF3 as a control (lane 1). Fluorescent IIIabc RNAwas used at 20 nM in concentration, and the reactions were carried out in the presence of 2μM tRNA to prevent
nonspecific binding. (B) Native agarose gel showing binding of the PCI/MPN octamer to the 40S ribosomal subunit, monitored by UV absorbance of the 40S
subunit rRNA. The nanomolar concentrations of the PCI/MPN octamer are given, and the 40S ribosomal subunit was used at a concentration of 10 nM. Shown as
a control, 40S subunit binding to natively purified human eIF3 (lane 1). (C) Coomassie blue-stained SDS gel showing PCI/MPN octamer affinity-purified using
MBP-tagged eIF1A, or using MBP-tagged eIF1. MW markers are shown in kilodaltons. Arrows indicate the position of MBP-eIF1A (lanes 2, 4) and MBP-eIF1
(lanes 5, 7). Binding andwash conditions prevent nonspecific binding of the PCI/MPN octamer tothe beads (lane 8). (D)Coomassie blue-stained SDS gel showing
reconstituted eIF3 10-mer affinity-purified using the FLAG-tagged central domain of eIF4G. MW markers are shown to the left. Arrow indicates the position of
subunit eIF3b. GroE copurified with eIF3d (asterisks), as determined by MS analysis. Antibody heavy (H) and light (L) chains are marked. The concentration of
KCl used in the washes (200 mM) prevents nonspecific binding to the anti-FLAG beads (Fig. S4).
Binding of eIF3 subassemblies to the HCV IRES IIIabc domain. (A) Native agarose gel showing binding of the PCI/MPN octamer to the HCV IRES IIIabc
recombinant eIF3 dodecamer. (A) Sucrose gradient of RRL translation reac-
tion stalled with GMPPNP and programmed with fluorescently labeled
HCV IRES-containing mRNA. The top of the gradient is to the left, and the
A254absorbance is shown. The break marked on the absorbance axis corre-
sponds to a fivefold decrease of sensitivity to account for heme absorbance.
Fractions pooled for GST affinity purification and analysis are marked F1-F4,
with F3 corresponding to HCV IRES-mediated initiation complexes (arrow).
(B) Western blotting of GST-eIF3d and eIF2α, from samples GST-affinity-pur-
ified from pooled fractions F1-F4 in A. (C) Native agarose gel of fluorescently
labeled HCV IRES copurified with GST-tagged eIF3 affinity-purified from
pooled fractions F1-F4 in A. (D) Northern blotting of 18S rRNA and tRNAi
present in GST-affinity-purified complexes from pooled fractions F1-F4 in
A. In this experiment, recombinant eIF3 dodecamer must compete with en-
dogenous eIF3 to form initiation complexes.
In vitro translation initiation complex formation in the presence of
plexes. (A) Negative-stain EM reconstructions of reconstituted 12-subunit
eIF3 and PCI/MPN octamer at resolutions of 29 and 23 Å, respectively. Both
reconstructions are shown in the same orientations, and are compared to the
cryoEM reconstruction of native human eIF3 (24), to the right. (B) Difference
map comparing the 12-subunit complex to the PCI/MPN octamer, filtered to a
resolution of 29 Å, and contoured at 5.4 sigma. Positive density is shown in
gold, and negative density in gray. (C) Interactions of the eIF3 PCI/MPN oc-
tamer with the remaining eIF3 subunits, with identified binary interactions
noted. No geometrical constraints on positioning are implied by the location
of the connecting lines.
Structural analysis of the PCI/MPN octamer and eIF3 dodecamer com-
Sun et al.PNAS
December 20, 2011
eight of these subunits are coexpressed in E. coli, forming a PCI/
MPN octamer (Fig. 1C). This PCI/MPN octamer is distinct from
the three modules identified by disassembly experiments using
mass spectrometry (3), indicating that the spontaneous assembly
of eIF3follows a different pathway thansalt-dependent disassem-
bly. Many of the interactions observed here have not been mod-
eled in the PCI/MPN cores of the proteasome lid or COP9
signalosome (1, 2, 4, 5), possibly due to the challenge of identify-
ing direct orthologues among the subunits in each complex. The
results obtained here may aid in identifying the phylogenetic
relationship between corresponding subunits in these cores (1).
From the PCI/MPN octamer as a starting point, we were able
to add subunits eIF3d, eIF3b, the eIF3g/eIF3i dimer, and eIF3j
serially to form an intact 700 kDa molecular mass human eIF3,
missing only two domains from subunits eIF3a and eIF3c
(Fig. 1A). Attempts to assemble a preformed bgi trimer with
the PCI/MPN octamer were not successful in our hands. Further-
more, binding of the eIF4G central domain to the 10-subunit
eIF3 complex destabilized eIF3b association with the reconsti-
tuted eIF3 complex (Fig. 2D, Fig. S4), an effect not seen with
natively purified eIF3. Whether b, g, and i, which are universally
conserved in eukaryotes, bind the preassembled PCI/MPN octa-
mer in a stepwise manner in vivo, or require chaperones for as-
sembly into eIF3,remains to bedetermined. Notably, a functional
6-subunit complex assembled in insect cells lacked three of the
PCI/MPN core proteins (22). This 6-subunit complex may have
been stabilized by the presence of subunit eIF3b, indicating that
interactions within the PCI/MPN core may be further stabilized
by the remaining subunits (b, d, g, i, and j).
The striking resemblance of the PCI/MPN octamer to intact
human eIF3 (Fig. 4) (24) suggests that eIF3 may envelope the
40S ribosomal subunit, using flexible regions emanating from
the PCI/MPN core in the assembly of preinitiation and initiation
complexes (24, 37–40). Genetic and biochemical experiments
identified interactions between subunits eIF3a and eIF3c and
the solvent side of the 40S subunit (37, 38). The flexible C ter-
minus of eIF3a is located near the mRNA entry tunnel, whereas
the N terminus of eIF3c is likely positioned near the 40S subunit
platform where it interacts with eIF1 and eIF5 situated at the
subunit interface (41). In addition, biochemical experiments
place the C terminus of eIF3j on the 60S subunit interface side
of the 40S subunit within the mRNA decoding site (39). Subunit
eIF3j, which is involved in regulating mRNA binding and posi-
tioning on the 40S ribosomal subunit during translation initiation
(39, 40), interacts with at least five subunits within human eIF3,
including four of the PCI/MPN domain-containing subunits (c, f,
h, and k) (Fig. 1, Fig. S1) (36). The only possible means for eIF3j
to approach the decoding site in the 40S subunit is for eIF3 to
nearly encircle the 40S subunit mRNA binding channel. Further-
more, models of the 40S subunit-eIF3 binary complex place one
leg of eIF3 in the way of the 60S subunit binding surface of the
40S subunit platform domain (24). The evidence presented here
shows that elements of the PCI/MPN core constitute this leg fea-
ture of eIF3. Finally, the PCI/MPN octamer interacts with eIF1
and eIF1A, both of which reside at the subunit interface during
initiation (31, 42–44), indicating that even the PCI/MPN core
identified here contains flexible domains (Fig. 5), in addition to
subunits a and c (Fig. 1A), and the flexible attachment of subunits
b, d, g, i, and j (Fig. 4B). Future efforts will be required to test the
functional role of the PCI/MPN core in aiding a highly flexible
eIF3 in the assembly of translation preinitiation and initiation
complexes, and whether the proteasome lid and COP9 signalo-
some also exploit this architectural strategy for functional inter-
actions with their cellular targets (45, 46).
Materials and Methods
Purification of eIF3, 40S Ribosomal Subunits and HCV IRES RNAs. Human eIF3
and 40S ribosomal subunits were purified from HeLa cell lysates as described
in refs. 9 and 25. Subunit eIF3j was expressed and purified as described in
ref. 39. HCV IRES RNA and the IRES IIIabc domain were transcribed and pur-
ified as described in ref. 47. The sequence of the IRES IIIabc domain is that
used in ref. 25. The IRES sequence used in RRL initiation complex formation
includes nucleotides 39–352 of the HCV subtype 1b genomic RNA.
In Vitro Reconstruction of eIF3 Subcomplexes. All eIF3 subunits were PCR
amplified from human cDNA (American Type Culture Collection) or were
codon-optimized and inserted into transfer vectors 2AT, 2CT, 2GT, 2ST, and
2XT (SI Materials and Methods, Table S1, Table S2) using ligation-indepen-
dent cloning (48, 49). The open reading frames for subunits eIF3b (N termi-
nus), eIF3e, eIF3i, and eIF3j were codon-optimized by DNA 2.0 (SI Materials
and Methods, Table S3). Vectors were developed by the MacroLab at the QB3
Institute, University of California, Berkeley, and are available from Addgene
(www.addgene.org). The preparation of transfer vectors containing eIF3
subunits and of polycistronic expression vectors is described in SI Materials
Expression and Purification of eIF3 Subcomplexes. All eIF3 subcomplexes
were expressed in Rosetta2(DE3)pLysS E. coli (EMD Biosciences), as described
in the SI Materials and Methods and Table S3. Individual subunits of eIF3 ex-
pressed with N-terminal fusion tags (Table S1, Table S2) were purified by nick-
el affinity chromatography [His6-Saccharomyces cerevisiae small ubiquitin-
like modifier (SUMO) and His6-γ-crystallin tags], glutathione affinity chroma-
tography (His6-GST tag), or dextrin sepharose affinity chromatography [His6-
E. coli maltose binding protein (MBP) tag], as described in SI Materials and
N-terminal tags were cleaved by tobacco etch protease (TEV) and the
cleaved tag and TEV protease were removed from the subunits and
complexes by passing the solution through the HisTrap HP column. The
flow-through fractions were then passed through a HiTrap Q column (GE
Healthcare) to remove nonspecifically bound RNA. Further details of the pur-
ification of different complexes are included in SI Materials and Methods.
EM Sample Preparation and Data Collection. Samples diluted to a final concen-
tration of 50 nM were placed onto continuous carbon grids, negatively
stained with a 3% uranyl acetate solution and blotted dry. Data were
acquired using a Tecnai T12 electron microscope equipped with a Tietz
4 × 4 Kpixel CCD camera using low dose techniques. All the processing of
two-dimensional data was performed using programs and utilities contained
within the Appion processing environment (50). Further details of image
acquisition, particle extraction, and generation of class averages is included
in SI Materials and Methods. The particles included in the final set of class
averages (21,472 and 12,452 particles for the PCI/MPN octamer and dodeca-
mer stacks, respectively) were generated from phase-flipped micrographs
using the estimated parameters from the CTFFind program (51). An initial
model of human eIF3 (24) filtered at 120-Å resolution was used for three-
dimensional refinement, performed using iterative projection matching in
EMAN2 (52, 53). Further details of volume analysis and difference map
calculation are included in SI Materials and Methods.
unit, eIF3j, and initiation factors eIF1 and eIF1A. The position of the PCI/MPN
octamer relative to the 40S subunit is based on that in ref. 24. The positions of
eIF1 and eIF1A are based on the X-ray crystal structure of the eIF1/40S com-
plex (44), and directed hydroxyl radical probing experiments of eIF1A bound
to the 40S subunit (43), or eIF3j bound to the 40S subunit (39). Inferred flex-
ible regions of the translation factors are indicated by dashed lines (eIF1 or
eIF1A) or a solid line (eIF3j), as well as an oval for regions of the PCI/MPN
octamer flexibly attached to the ordered core.
Model for interactions of the eIF3 PCI/MPN octamer with the 40S sub-
www.pnas.org/cgi/doi/10.1073/pnas.1116821108Sun et al.
The 40S Ribosomal Subunit-eIF3 (Sub)complex Formation. Binding reactions
were carried out at 25°C for 15 min in ribosome binding buffer (20 mM
Hepes, pH ¼ 7.5, 100 mM KCl, 2.5 mM MgCl2). Native agarose gel electro-
phoresis used to monitor binding was carried out at 4°C in buffer containing
34 mM Tris, 66 mM Hepes, 0.1 mM EDTA, 2.5 mM MgCl2, 75 mM KCl, pH ¼ 7.8,
hereafter termed THEMK buffer, with frequent buffer exchanges.
eIF3 (Sub)complex Interactions with Labeled IIIabc HCV IRES and eif3j-Alexa488.
Binding reactions to the IIIabc HCV IRES domain contained reconstituted
human eIF3, labeled HCV IRES IIIabc domain (SI Materials and Methods)
at 20 nM, and THEMK buffer. The reactions were carried out at 25°C for
15 min, then resolved by native 1% agarose gels containing THEMK buffer,
at 4 °C for 45 min. Similarly, binding of eIF3 subcomplexes to labeled eIF3j
(SI Materials and Methods) was assayed by native agarose gels containing
THEMK buffer. The resulting agarose gels were analyzed by fluorescence
imaging using a Typhoon Scanner (Amersham Biosciences) to detect the
gel shifts. The excitation and emission wavelengths to detect the HCV IRES
IIIabc domain and eIF3j were 494 and 518 nm, respectively.
eIF1, eIF1A, and eIF4G Pull-Down Assays. For eIF1A or eIF1 pull-down assays,
50 μL of amylose resin (New England BioLabs) was washed three times with
MBP column buffer A (20 mM Hepes, pH 1/4 7.5, 300 mM KCl, 1 mM EDTA,
1 mM DTT, 10% glycerol) and then incubated with 20 μg of purified His6-
MBP-eIF1A or His6-MBP-eIF1 (SI Materials and Methods) for 1 h at 4°C with
gentle rotation. The beads were pelleted, to allow for removal of the super-
natant. Following three washes with MBP column buffer A, 100 μg of purified
PCI/MPN octamer was incubated with the beads for 2 h at 4°C. After remov-
ing the supernatant and washing the beads three times, bound proteins were
eluted using MBP column buffer B (20 mM Hepes, pH 1/4 7.5, 300 mM KCl,
10 mM maltose, 1 mM EDTA, 1 mM DTT, 10% glycerol).
A truncated version of human eIF4G (amino acids 1011–1104) with an
N-terminal His6-FLAG tag (His6-FLAG-eIF4Gt) was used for eIF4G pull-down
assays. Pull-down assays were carried out using resin linked to anti-FLAG anti-
bodies (ANTIFLAG M2 affinity gel, Sigma). Briefly, 60 μL of resin slurry were
washed three times with 500 μL of dilution buffer (20 mM Hepes pH ¼ 7.5,
150 mM KCl, 10% glycerol), followed by addition of 10 μg of His6-FLAG-
eIF4Gt to allow the FLAG-tagged truncated eIF4G to bind to the resin.
The resin was then washed three times with 100 μL wash buffer (20 mM
Hepes pH ¼ 7.5, 200 mM KCl, 0.5% Triton X-100, 0.5 mM DTT, 5% glycerol).
Natively purified eIF3 or reconstituted eIF3 complexes were then added to
the resin and incubated with the resin for 30 min on ice. The resin was then
washed three times with wash buffer to remove unbound protein. Any
bound eIF3/eIF4G complex was then eluted by adding 7.5 μg FLAG peptide
in dilution buffer.
In Vitro Translation Assays of Reconstituted eIF3. The purification of the 12-
subunit eIF3-containing GST-tagged eIF3d was as described in SI Materials
and Methods, but with the GST tag retained prior to the final gel filtration
step. In vitro translation assays were carried out using nuclease-treated
RRL (Promega), essentially as described in refs. 54 and 55. Details of the trans-
lation assays are given in SI Materials and Methods. Gradients of the RRL
reactions were fractionated using an ISCO UV detector and 0.33 mL fractions
were collected for subsequent native agarose gel and affinity purification
To track the HCV IRES, 10 μL of each fraction was resolved by native
agarose gel electrophoresis in THEMK buffer, as described above. Fractions
from the sucrose gradients were pooled into four larger fractions (F1-F4), to
which additional RNasin Plus RNase Inhibitor and Complete Protease Inhibi-
tor Cocktail were added. The four fractions were then dialyzed into THEMK
buffer for 2 h at 4°C. To affinity purify complexes containing GST-tagged
eIF3, the pooled fractions were mixed gently with 0.1 mL reduced Glu-
tathione-Sepharose 4B beads (GE Healthcare) for 2 hr at 4°C. The beads were
collected by brief centrifugation and washed three times with THEMK buffer.
Proteins bound to the beads were eluted with THEMK buffer containing
10 mM reduced glutathione.
The affinity-purified complexes were then analyzed by Western and
Northern blotting, as follows. The presence of GST-eIF3d was probed with
an anti-GST antibody (Abcam), as described in ref. 56. Similarly, the presence
of eIF2 was probed with an anti-eIF2α antibody (Cell Signaling), and subunit
eIF3a or its truncated version (a*) was probed using an anti-eIF3a antibody
(Santa Cruz). To probe for the 40S ribosomal subunit and tRNAi, an 18S rRNA-
specific probe (5′-ACGGTATCTGATCGTCTTCGAACC-3′) (57) and a tRNAi-
specific probe (5′-TGGTAGCAGAGGATGGTTTCGAT-3′) were used. The probes
were labeled on the 5′ end with [γ-32P] ATP (Perkin Elmer) using T4 polynu-
cleotide kinase (New England BioLabs). Details of the Northern blotting
analyses are given in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Robert Tjian for HeLa cell cytoplasmic
extracts, Anthony Iavarone for help with mass spectrometry data acquisition
and interpretation, Masaaki Sokabe for help with protocols for Northern
analyses, and John Hershey for helpful comments on the manuscript.
This work was funded by the National Institutes of Health Grant
P01GM073732 (to J.H.D.C. and J.A.D.), R01GM092927 (to C.S.F.), and
1S10RR022393-01 to the QB3/Department of Chemistry Mass Spectrometry
facility. J.A.D. and E.N. are Howard Hughes Medical Institute Investigators.
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