Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

Laboratory of Regulation of Gene Expression Institute of Microbiology ASCR v v i Videnska 1083 Prague 142 20 Czech Republic

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Hinnebusch AG. eIF3: a versatile scaffold for translation initiation complexes. Trends Biochem Sci. 2006;31:553–562. PubMed

Jackson RJ, Hellen CUT, Pestova TV. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 2010;11:113–127. PubMed PMC

Phan L, Schoenfeld LW, Valášek L, Nielsen KH, Hinnebusch AG. A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNAiMet. EMBO J. 2001;20:2954–2965. PubMed PMC

Naranda T, Kainuma M, McMillan SE, Hershey JWB. The 39-kilodalton subunit of eukaryotic translation initiation factor 3 is essential for the complex's integrity and for cell viability in Saccharomyces cerevisiae. Mol. Cell. Biol. 1997;17:145–153. PubMed PMC

Cuchalová L, Kouba T, Herrmannová A, Danyi I, Chiu W-l, Valášek L. The RNA recognition motif of eukaryotic translation initiation factor 3g (eIF3g) is required for resumption of scanning of posttermination ribosomes for reinitiation on GCN4 and together with eIF3i stimulates linear scanning. Mol. Cell. Biol. 2010;30:4671–4686. PubMed PMC

Masutani M, Sonenberg N, Yokoyama S, Imataka H. Reconstitution reveals the functional core of mammalian eIF3. EMBO J. 2007;26:3373–3383. PubMed PMC

Fraser CS, Lee JY, Mayeur GL, Bushell M, Doudna JA, Hershey JW. The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40S ribosomal subunits in vitro. J. Biol. Chem. 2004;279:8946–8956. PubMed

Zhou M, Sandercock AM, Fraser CS, Ridlova G, Stephens E, Schenauer MR, Yokoi-Fong T, Barsky D, Leary JA, Hershey JW, et al. Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3. Proc. Natl Acad. Sci. USA. 2008;105:18139–18144. PubMed PMC

Valášek L, Nielsen KH, Hinnebusch AG. Direct eIF2-eIF3 contact in the multifactor complex is important for translation initiation in vivo. EMBO J. 2002;21:5886–5898. PubMed PMC

Asano K, Phan L, Anderson J, Hinnebusch AG. Complex formation by all five homologues of mammalian translation initiation factor 3 subunits from yeast Saccharomyces cerevisiae. J. Biol. Chem. 1998;273:18573–18585. PubMed

ElAntak L, Tzakos AG, Locker N, Lukavsky PJ. Structure of eIF3b RNA recognition motif and its interaction with eIF3j: structural insights into the recruitment of eIF3b to the 40 S ribosomal subunit. J. Biol. Chem. 2007;282:8165–8174. PubMed

ElAntak L, Wagner S, Herrmannová A, Karásková M, Rutkai E, Lukavsky PJ, Valášek L. The indispensable N-terminal half of eIF3j co-operates with its structurally conserved binding partner eIF3b-RRM and eIF1A in stringent AUG selection. J. Mol. Biol. 2010;396:1097–1116. PubMed PMC

Valášek L, Phan L, Schoenfeld LW, Valášková V, Hinnebusch AG. Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in PRT1 required for eIF3 integrity and ribosome binding. EMBO J. 2001;20:891–904. PubMed PMC

Marintchev A, Wagner G. Translation initiation: structures, mechanisms and evolution. Q. Rev. Biophys. 2005;37:197–284. PubMed

Dodd RB, Allen MD, Brown SE, Sanderson CM, Duncan LM, Lehner PJ, Bycroft M, Read RJ. Solution structure of the Kaposi's sarcoma-associated Herpesvirus K3 N-terminal domain reveals a novel E2-binding C4HC3-type RING domain. J. Biol. Chem. 2004;279:53840–53847. PubMed

Muchmore SW, Sattler M, Liang H, Meadows RP, Harlan JE, Yoon HS, Nettesheim D, Chang BS, Thompson CB, Wong S-L, et al. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 1996;381:335–341. PubMed

Szymczyna BR, Taurog RE, Young MJ, Snyder JC, Johnson JE, Williamson JR. Synergy of NMR, computation, and X-ray crystallography for structural biology. Structure. 2009;17:499–507. PubMed PMC

Leslie A. The integration of macromolecular diffraction data. Acta Cryst.Section D. 2006;62:48–57. PubMed

Evans P. Scaling and assessment of data quality. Acta Cryst. Section D. 2006;62:72–82. PubMed

French S, Wilson K. On the treatment of negative intensity observations. Acta Cryst Section A. 1978;34:517–525.

Collaborative Computational Project N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol Crystallogr. 1994;50:760–763. PubMed

de La Fortelle E, Bricogne G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 1997;276:472–494. PubMed

Kelley LA, Sternberg MJE. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protocols. 2009;4:363–371. PubMed

McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ. Likelihood-enhanced fast translation functions. Acta Cryst. Section D. 2005;61:458–464. PubMed

Zhang KY, Cowtan K, Main P. Combining constraints for electron-density modification. Methods Enzymol. 1997;277:53–64. PubMed

Cowtan K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Cryst. Section D. 2006;62:1002–1011. PubMed

Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Cryst. Section D. 2004;60:2126–2132. PubMed

Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Cryst. Section D. 1997;53:240–255. PubMed

Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW, Arendall WB, III, Snoeyink J, Richardson JS, et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 2007;35:W375–383. PubMed PMC

Coyle SM, Gilbert WV, Doudna JA. Direct Link between RACK1 function and localization at the ribosome in vivo. Mol. Cell. Biol. 2009;29:1626–1634. PubMed PMC

Li D, Roberts R. Human genome and diseases: WD-repeat proteins: structure characteristics, biological function, and their involvement in human diseases. Cell. Mol. Life Sci. 2001;58:2085–2097. PubMed PMC

Jennings BH, Pickles LM, Wainwright SM, Roe SM, Pearl LH, Ish-Horowicz D. Molecular recognition of transcriptional repressor motifs by the WD domain of the groucho/TLE corepressor. Mol. Cell. 2006;22:645–655. PubMed

Oliver AW, Swift S, Lord CJ, Ashworth A, Pearl LH. Structural basis for recruitment of BRCA2 by PALB2. EMBO Rep. 2009;10:990–996. PubMed PMC

ter Haar E, Harrison SC, Kirchhausen T. Peptide-in-groove interactions link target proteins to the Î2-propeller of clathrin. Proc. Natl Acad. Sci. USA. 2000;97:1096–1100. PubMed PMC

Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 2007;372:774–797. PubMed

Gallivan JP, Dougherty DA. Cation-pi interactions in structural biology. Proc. Natl Acad. Sci. USA. 1999;96:9459–9464. PubMed PMC

Valášek L, Mathew A, Shin BS, Nielsen KH, Szamecz B, Hinnebusch AG. The yeast eIF3 subunits TIF32/a and NIP1/c and eIF5 make critical connections with the 40S ribosome in vivo. Genes Dev. 2003;17:786–799. PubMed PMC

Valášek L, Szamecz B, Hinnebusch AG, Nielsen KH. In vivo stabilization of preinitiation complexes by formaldehyde cross-linking. Methods Enzymol. 2007;429:163–183. PubMed

Valášek L, Nielsen KH, Zhang F, Fekete CA, Hinnebusch AG. Interactions of eukaryotic translation initiation factor 3 (eIF3) subunit NIP1/c with eIF1 and eIF5 promote preinitiation complex assembly and regulate start codon selection. Mol. Cell. Biol. 2004;24:9437–9455. PubMed PMC

Fekete CA, Mitchell SF, Cherkasova VA, Applefield D, Algire MA, Maag D, Saini AK, Lorsch JR, Hinnebusch AG. N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection. EMBO J. 2007;26:1602–1614. PubMed PMC

Nanda JS, Cheung Y-N, Takacs JE, Martin-Marcos P, Saini AK, Hinnebusch AG, Lorsch JR. eIF1 controls multiple steps in start codon recognition during eukaryotic translation initiation. J. Mol. Biol. 2009;394:268–285. PubMed PMC

Hinnebusch AG. Translational regulation of GCN4 and the general amino acid control of yeast. Annu. Rev. Microbiol. 2005;59:407–450. PubMed

Nielsen KH, Szamecz B, Valasek L, Jivotoskaya A, Shin BS, Hinnebusch AG. Functions of eIF3 downstream of 48S assembly impact AUG recognition and GCN4 translational control. EMBO J. 2004;23:1166–1177. PubMed PMC

Szamecz B, Rutkai E, Cuchalova L, Munzarova V, Herrmannova A, Nielsen KH, Burela L, Hinnebusch AG, Valášek L. eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA. Genes Dev. 2008;22:2414–2425. PubMed PMC

Munzarová V, Pánek J, Gunišová S, Dányi I, Szamecz B, Valášek LS. Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs. PLoS Genet. 2011;7:e1002137. PubMed PMC

Mitchell SF, Lorsch JR. Should I stay or should i go? Eukaryotic translation initiation factors 1 and 1a control start codon recognition. J. Biol. Chem. 2008;283:27345–27349. PubMed PMC

Cheung YN, Maag D, Mitchell SF, Fekete CA, Algire MA, Takacs JE, Shirokikh N, Pestova T, Lorsch JR, Hinnebusch AG. Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo. Genes Dev. 2007;21:1217–1230. PubMed PMC

Chiu W-L, Wagner S, Herrmannova A, Burela L, Zhang F, Saini AK, Valasek L, Hinnebusch AG. The C-terminal region of eukaryotic translation initiation factor 3a (eIF3a) promotes mRNA recruitment, scanning, and, together with eIF3j and the eIF3b RNA recognition motif, selection of AUG start codons. Mol. Cell. Biol. 2010;30:4415–4434. PubMed PMC

Passmore LA, Schmeing TM, Maag D, Applefield DJ, Acker MG, Algire MA, Lorsch JR, Ramakrishnan V. The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome. Mol. Cell. 2007;26:41–50. PubMed

Mitchell SF, Walker SE, Algire MA, Park E-H, Hinnebusch AG, Lorsch JR. The 5′-7-Methylguanosine Cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway. Mol. Cell. 2010;39:950–962. PubMed PMC

Jivotovskaya A, Valášek L, Hinnebusch AG, Nielsen KH. Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast. Mol. Cell. Biol. 2006;26:1355–1372. PubMed PMC

Pisarev AV, Kolupaeva VG, Yusupov MM, Hellen CUT, Pestova TV. Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes. EMBO J. 2008;27:1609–1621. PubMed PMC

Saini AK, Nanda JS, Lorsch JR, Hinnebusch AG. Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNAiMet binding to the ribosome. Genes Dev. 2010;24:97–110. PubMed PMC

Pestova TV, Borukhov SI, Hellen CUT. Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons. Nature. 1998;394:854–859. PubMed

Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA. Electrostatics of nanosystems: Application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA. 2001;98:10037–10041. PubMed PMC

Dolinsky TJ, Czodrowski P, Li H, Nielsen JE, Jensen JH, Klebe G, Baker NA. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res. 2007;35:W522–W525. PubMed PMC

Taylor DJ, Devkota B, Huang AD, Topf M, Narayanan E, Sali A, Harvey SC, Frank J. Comprehensive molecular structure of the eukaryotic ribosome. Structure. 2009;17:1591–1604. PubMed PMC

Kuzmic P. DynaFit--a software package for enzymology. Methods in Enzymology. 2009;467:247–280. PubMed

Kuzmic P. Program DYNAFIT for the analysis of enzyme kinetic data: Application to HIV proteinase. Analyt. Biochem. 1996;237:260–273. PubMed

Nielsen KH, Valášek L. In vivo deletion analysis of the architecture of a multi-protein complex of translation initiation factors. Methods Enzymol. 2007;431:15–32. PubMed

Grant CM, Miller PF, Hinnebusch AG. Requirements for intercistronic distance and level of eIF-2 activity in reinitiation on GCN4 mRNA varies with the downstream cistron. Mol. Cell. Biol. 1994;14:2616–2628. PubMed PMC

Cigan AM, Foiani M, Hannig EM, Hinnebusch AG. Complex formation by positive and negative translational regulators of GCN4. Mol. Cell. Biol. 1991;11:3217–3228. PubMed PMC

Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988;67:31–40. PubMed

Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988;74:527–534. PubMed

Adapted formaldehyde gradient cross-linking protocol implicates human eIF3d and eIF3c, k and l subunits in the 43S and 48S pre-initiation complex assembly, respectively

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle

Human eIF3b and eIF3a serve as the nucleation core for the assembly of eIF3 into two interconnected modules: the yeast-like core and the octamer

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

Translation initiation factor eIF3 promotes programmed stop codon readthrough

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

Translation initiation factors eIF3 and HCR1 control translation termination and stop codon read-through in yeast cells

Functional characterization of the role of the N-terminal domain of the c/Nip1 subunit of eukaryotic initiation factor 3 (eIF3) in AUG recognition

Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit

'Ribozoomin'--translation initiation from the perspective of the ribosome-bound eukaryotic initiation factors (eIFs)

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PDB
3ZWL