Novel insights into the architecture and protein interaction network of yeast eIF3

Abstract

Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs). The largest of these factors, eIF3, forms the scaffold for other initiation factors, promoting their binding to the 40S ribosomal subunit. Biochemical and structural studies on eIF3 need highly pure eIF3. However, natively purified eIF3 comprise complexes containing other proteins such as eIF5. Therefore we have established in vitro reconstitution protocols for Saccharomyces cerevisiae eIF3 using its five recombinantly expressed and purified subunits. This reconstituted eIF3 complex (eIF3 rec ) exhibits the same size and activity as the natively purified eIF3 (eIF3 nat ). The homogeneity and stoichiometry of eIF3 rec and eIF3 nat were confirmed by analytical size exclusion chromatography, mass spectrometry, and multi-angle light scattering, demonstrating the presence of one copy of each subunit in the eIF3 complex. The reconstituted and native eIF3 complexes were compared by single-particle electron microscopy showing a high degree of structural conservation. The interaction network between eIF3 proteins was studied by means of limited proteolysis, analytical size exclusion chromatography, in vitro binding assays, and isothermal titration calorimetry, unveiling distinct protein domains and subcomplexes that are critical for the integrity of the protein network in yeast eIF3. Taken together, the data presented here provide a novel procedure to obtain highly pure yeast eIF3, suitable for biochemical and structural analysis, in addition to a detailed picture of the network of protein interactions within this complex.

Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes

“…As expected (59), no binding was detected between b/PRT1 and a/TIF32 276 − 494 as judged by no change in the retention volumes of the mixture of the two proteins on analytical SEC compared with those of each protein alone (Figure 5C). Hence, the binding of a/TIF32 to b/PRT1 is most likely mediated via the C-terminal part of a/TIF32, as we recently also showed by in vitro reconstitution experiments (45).

Figure 5.Protein and RNA-binding properties of a/TIF32 276-494 .

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Novel RNA-binding Protein P311 Binds Eukaryotic Translation Initiation Factor 3 Subunit b (eIF3b) to Promote Translation of Transforming Growth Factor β1-3 (TGF-β1-3)

“…Several of them have been identified as core subunits (47). eIF3b is one of them, whereas the others are eIF3j, eIF3a, eIF3g, eIF3i, and eIF3e (25,48). These 68 aa).…”

Mechanism of Cytoplasmic mRNA Translation

“…The remaining subunits, b, d and g have RNA Recognition Motif (RRM) domains (Cuchalova et al, 2010); subunits b and i have WD40 domains and eIF3g has a zinc-binding domain Valasek, 2012;Voigts-Hoffmann et al, 2012;Hashem et al, 2013;Querol-Audi et al, 2013). Recent cryo-EM reconstructions of mammalian eIF3 and the 43S PIC indicate that eIF3 has five lobes and the PCI/MPN octamer forms the functional core of eIF3 (Siridechadilok et al, 2005;Khoshnevis et al, 2012;Hashem et al, 2013;Querol-Audi et al, 2013). The placement of the eIF3 subunits and their contacts with the ribosome and other initiation factors awaits further structural data, but a picture is beginning to emerge at the molecular level (Wilson and Doudna Cate, 2012;Hashem et al, 2013;Liu et al, 2014).…”