Subassemblies of eIF3 expressed in E. coli. (A) Truncations in subunits 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 nonamer (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) expressed 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 incubated with other recombinant eIF3 components (lanes 2–5), or native human eIF3 depleted of eIF3j (lane 6).

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 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 RNA was used at 20 nM in concentration, and the reactions were carried out in the presence of 2 μM 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 and wash conditions prevent nonspecific binding of the PCI/MPN octamer to the 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).

In vitro translation initiation complex formation in the presence of recombinant eIF3 dodecamer. (A) Sucrose gradient of RRL translation reaction stalled with GMPPNP and programmed with fluorescently labeled HCV IRES-containing mRNA. The top of the gradient is to the left, and the A₂₅₄ absorbance is shown. The break marked on the absorbance axis corresponds 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-purified 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 tRNA_i present in GST-affinity-purified complexes from pooled fractions F1-F4 in A. In this experiment, recombinant eIF3 dodecamer must compete with endogenous eIF3 to form initiation complexes.

Structural analysis of the PCI/MPN octamer and eIF3 dodecamer complexes. (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 octamer with the remaining eIF3 subunits, with identified binary interactions noted. No geometrical constraints on positioning are implied by the location of the connecting lines.

Model for interactions of the eIF3 PCI/MPN octamer with the 40S subunit, 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 complex (44), and directed hydroxyl radical probing experiments of eIF1A bound to the 40S subunit (43), or eIF3j bound to the 40S subunit (39). Inferred flexible 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.