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

Sun, Chaomin; Todorović, Aleksandar; Querol-Audí, Jordi; Bai, Yun; Villa, Nancy Y.; Snyder, Monica; Ashchyan, John; Lewis, Christopher S.; Hartland, Abbey; Gradia, Scott; Fraser, Christopher S.; Doudna, Jennifer A.; Nogales, Eva; Cate, J.H.D.

doi:10.1073/pnas.1116821108

Cited by 103 publications

(180 citation statements)

References 57 publications

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“…This proposed model of 40S binding mode of eIF3 is in accordance with that available for yeast (Valasek et al 2003). A recent study on the reconstitution of human eIF3 has revealed that an octameric subcomplex of eIF3 comprising subunits e, f, h, k, l, and m and truncated fragments of subunits a and c exhibits a striking structural resemblance to the previously determined cryo-EM structure of native human eIF3 (Sun et al 2011).…”

Section: Introductionsupporting

confidence: 56%

“…Such a reconstitution system would, on the one hand, provide new strategies for a detailed kinetic and thermodynamic dissection of translation initiation in eukaryotes and, on the other hand, facilitate structural studies of eIF3 and its interaction partners. Recently, Sun et al (2011) described the reconstitution of human eIF3 using recombinantly expressed and purified subcomplexes. Using this system, the importance of an octameric subcomplex of eIF3 for binding to the 40S ribosomal subunit and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) was demonstrated (Sun et al 2011).…”

Section: Introductionmentioning

confidence: 99%

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

Khoshnevis

Hauer

Milón

et al. 2012

RNA

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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.

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Section: Introductionsupporting

confidence: 56%

Section: Introductionmentioning

confidence: 99%

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

Khoshnevis

Hauer

Milón

et al. 2012

RNA

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“…[119]. Intriguingly, eIF3 is still capable of binding the IRES after being subjected to limited proteolysis [120], and a reconstituted octamer of eIF3 (a, c, e, f, h, k, l, m) is capable of high-affinity binding to an RNA fragment containing domain IIIa-c [121]. Although a full eIF3 complex appears to be important for efficiently forming 48S complexes [121], this suggests that IRES : eIF3 affinity does not require the full complex.…”

Section: (D) the Structural And Functional Basis Of Hcv Ires : Eif3 Imentioning

confidence: 99%

Dynamics of IRES-mediated translation

Johnson

Grosely

Petrov

et al. 2017

Phil. Trans. R. Soc. B

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One contribution of 12 to a theme issue 'Perspectives on the ribosome'. Viral internal ribosome entry sites (IRESs) are unique RNA elements, which use stable and dynamic RNA structures to recruit ribosomes and drive protein synthesis. IRESs overcome the high complexity of the canonical eukaryotic translation initiation pathway, often functioning with a limited set of eukaryotic initiation factors. The simplest types of IRESs are typified by the cricket paralysis virus intergenic region (CrPV IGR) and hepatitis C virus (HCV) IRESs, both of which independently form high-affinity complexes with the small (40S) ribosomal subunit and bypass the molecular processes of cap-binding and scanning. Owing to their simplicity and ribosomal affinity, the CrPV and HCV IRES have been important models for structural and functional studies of the eukaryotic ribosome during initiation, serving as excellent targets for recent technological breakthroughs in cryogenic electron microscopy (cryo-EM) and single-molecule analysis. High-resolution structural models of ribosome : IRES complexes, coupled with dynamics studies, have clarified decades of biochemical research and provided an outline of the conformational and compositional trajectory of the ribosome during initiation. Here we review recent progress in the study of HCV-and CrPV-type IRESs, highlighting important structural and dynamics insights and the synergy between cryo-EM and single-molecule studies.This article is part of the themed issue 'Perspectives on the ribosome'.

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“…The PCI subunits form a horseshoe-shaped structure and the MPN domains form a heterodimer, which are connected by a large helical bundle, to which all subunits contribute (13,17,18). Each of these eight subunits has paralogs in the COP9/signalosome (CSN) and the elongation initiation factor 3 (eIF3), which likely adopt a similar architecture (18)(19)(20)(21).The lid strengthens the interaction between the CP and RP (17) and deubiquitylates substrates before their processing by the AAA-ATPase module and the CP. Cleavage of polyubiquitin…”

mentioning

confidence: 99%

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

Pathare

Nagy

Śledź

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

120

131

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The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1-Pad1-N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins.n eukaryotes, the ubiquitin (Ub) proteasome system (UPS) is responsible for the regulated degradation of proteins (1-5). The UPS plays a key role in the maintenance of protein homeostasis by removing misfolded or damaged proteins, which could impair cellular functions, and by removing proteins whose functions are no longer needed. Consequently, the UPS is critically involved in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair, and malfunctions of the system often result in disease.The 26S proteasome executes the degradation of substrates that are marked for destruction by the covalent attachment of polyubiquitin chains. It is a molecular machine of 2.5 MDa comprising two subcomplexes, the 20S core particle (CP) and one or two 19S regulatory particles (RPs), which associate with the ends of the cylinder-shaped CP (6-8). The recognition and recruitment of polyubiquitylated substrates, their deubiquitylation, ATP-dependent unfolding, and translocation into the core particle take place in the RP. The structurally and mechanistically well-characterized CP houses the proteolytic activities and sequesters them from the environment, thereby avoiding collateral damage (9).The RPs attach to the outer α-rings of the CP, which control access to the proteolytic chamber formed by the inner β-subunit rings (10). Recently, the molecular architecture of the 26S holocomplex was established using cryo-EM-based approaches (11,12), and a pseudoatomic model of the holocomplex was put forward (13). The RP is formed by two subcomplexes, known as the base and the lid, which assemble independent...

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

Cited by 103 publications

References 57 publications

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

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

Dynamics of IRES-mediated translation

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

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