Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3)

California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2011; 108(51):20473-8. DOI: 10.1073/pnas.1116821108
Source: PubMed

ABSTRACT 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 structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent 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 reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.

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Available from: Christopher S Fraser, Mar 20, 2014
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    • "Furthermore, RACK1 was shown to associate with one of the eIF3 subunits in order to assemble a translation preinitiation complex in yeast (Hashem et al., 2013a; Kouba et al., 2012). Although our understanding of the molecular structure of the core of the 13 subunits eIF3 complex has progressed remarkably in recent years (e.g., Hashem et al., 2013b; Sun et al., 2011), the role of the noncore subunits remains essentially untested in an- imals. "
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    • "The helix interactions within the bundle appear to provide most of the energy for the assembly of lid subunits, whereas the weaker PCI-PCI interactions may primarily contribute specificity and thus determine the subunit order within the horseshoe-shaped arrangement of PCI subunits. This specificity contribution might be important considering that highly homologous PCI domain subunits are also present in other macromolecular complexes such as eIF3 and the CSN (Pick et al., 2009; Sun et al., 2011). The requirement of the bundle for lid assembly could thus prevent the incorporation of PCI-containing subunits from other complexes. "
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