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Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly.

Herrmannová A, Daujotyte D, Yang JC, Cuchalová L, Gorrec F, Wagner S, Dányi I, Lukavsky PJ, Valásek LS - Nucleic Acids Res. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

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The tif34-DD/KK mutation impairs the direct interaction between i/TIF34 and b/PRT1 in vitro and the revised 3D model of eIF3 in the MFC. (A) The prt1-W674A, -Y677A, and -R678A mutations impair the direct interaction between b/PRT1 and i/TIF34 in vitro. Full-length i/TIF34 (lane 3) and g/TIF35 (lane 4) fused to GST, and GST alone (lane 2), were tested for binding to 35S-labeled wt b/PRT1 and its mutant derivatives; 10% of input amounts added to each reaction is shown in lane 1 (In). (B) Full-length b/PRT1 (lane 3) and g/TIF35 (lane 4) fused to GST, and GST alone (lane 2), were tested for binding to 35S-labeled wt i/TIF34 and the DD/KK mutant derivative. (C) A revised 3D model of eIF3 and its associated eIFs in the MFC (based on the data from (9); ntd, N-terminal domain; ctd, C-terminal domain; hld, HCR1-like domain; rrm, RNA recognition motif; TC, ternary complex). The NMR structure of the interaction between the RRM of human eIF3b (green and light blue) and the N-terminal peptide of human eIF3j (yellow) (12), the NMR structure of the C-terminal RRM of human eIF3g (red and sky-blue) (5), and the X-ray structure of the yeast i/TIF34–b/PRT1 complex (this study), were used to replace the original schematic representations of the corresponding molecules.
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gkr765-F4: The tif34-DD/KK mutation impairs the direct interaction between i/TIF34 and b/PRT1 in vitro and the revised 3D model of eIF3 in the MFC. (A) The prt1-W674A, -Y677A, and -R678A mutations impair the direct interaction between b/PRT1 and i/TIF34 in vitro. Full-length i/TIF34 (lane 3) and g/TIF35 (lane 4) fused to GST, and GST alone (lane 2), were tested for binding to 35S-labeled wt b/PRT1 and its mutant derivatives; 10% of input amounts added to each reaction is shown in lane 1 (In). (B) Full-length b/PRT1 (lane 3) and g/TIF35 (lane 4) fused to GST, and GST alone (lane 2), were tested for binding to 35S-labeled wt i/TIF34 and the DD/KK mutant derivative. (C) A revised 3D model of eIF3 and its associated eIFs in the MFC (based on the data from (9); ntd, N-terminal domain; ctd, C-terminal domain; hld, HCR1-like domain; rrm, RNA recognition motif; TC, ternary complex). The NMR structure of the interaction between the RRM of human eIF3b (green and light blue) and the N-terminal peptide of human eIF3j (yellow) (12), the NMR structure of the C-terminal RRM of human eIF3g (red and sky-blue) (5), and the X-ray structure of the yeast i/TIF34–b/PRT1 complex (this study), were used to replace the original schematic representations of the corresponding molecules.

Mentions: To further test whether the mutations under study impair a direct contact between b/PRT1 and i/TIF34 proteins we introduced three single-Ala-substitution mutations into full length b/PRT1, synthesized and radiolabeled the resulting mutant proteins in vitro and tested their binding affinities towards i/TIF34 and g/TIF35 fused with the GST moiety. As shown in Figure 4A, all three completely eliminate binding of b/PRT1 specifically to i/TIF34 but not to g/TIF35. Similarly, both single D/K substitutions and the DD/KK double mutation of i/TIF34, the latter of which was chosen for further analysis, abolished binding of [35S]-labeled i/TIF34 to GST-b/PRT1 but not to GST-g/TIF35 (Figure 4B and data not shown).Figure 4.


Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly.

Herrmannová A, Daujotyte D, Yang JC, Cuchalová L, Gorrec F, Wagner S, Dányi I, Lukavsky PJ, Valásek LS - Nucleic Acids Res. (2011)

The tif34-DD/KK mutation impairs the direct interaction between i/TIF34 and b/PRT1 in vitro and the revised 3D model of eIF3 in the MFC. (A) The prt1-W674A, -Y677A, and -R678A mutations impair the direct interaction between b/PRT1 and i/TIF34 in vitro. Full-length i/TIF34 (lane 3) and g/TIF35 (lane 4) fused to GST, and GST alone (lane 2), were tested for binding to 35S-labeled wt b/PRT1 and its mutant derivatives; 10% of input amounts added to each reaction is shown in lane 1 (In). (B) Full-length b/PRT1 (lane 3) and g/TIF35 (lane 4) fused to GST, and GST alone (lane 2), were tested for binding to 35S-labeled wt i/TIF34 and the DD/KK mutant derivative. (C) A revised 3D model of eIF3 and its associated eIFs in the MFC (based on the data from (9); ntd, N-terminal domain; ctd, C-terminal domain; hld, HCR1-like domain; rrm, RNA recognition motif; TC, ternary complex). The NMR structure of the interaction between the RRM of human eIF3b (green and light blue) and the N-terminal peptide of human eIF3j (yellow) (12), the NMR structure of the C-terminal RRM of human eIF3g (red and sky-blue) (5), and the X-ray structure of the yeast i/TIF34–b/PRT1 complex (this study), were used to replace the original schematic representations of the corresponding molecules.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300007&req=5

gkr765-F4: The tif34-DD/KK mutation impairs the direct interaction between i/TIF34 and b/PRT1 in vitro and the revised 3D model of eIF3 in the MFC. (A) The prt1-W674A, -Y677A, and -R678A mutations impair the direct interaction between b/PRT1 and i/TIF34 in vitro. Full-length i/TIF34 (lane 3) and g/TIF35 (lane 4) fused to GST, and GST alone (lane 2), were tested for binding to 35S-labeled wt b/PRT1 and its mutant derivatives; 10% of input amounts added to each reaction is shown in lane 1 (In). (B) Full-length b/PRT1 (lane 3) and g/TIF35 (lane 4) fused to GST, and GST alone (lane 2), were tested for binding to 35S-labeled wt i/TIF34 and the DD/KK mutant derivative. (C) A revised 3D model of eIF3 and its associated eIFs in the MFC (based on the data from (9); ntd, N-terminal domain; ctd, C-terminal domain; hld, HCR1-like domain; rrm, RNA recognition motif; TC, ternary complex). The NMR structure of the interaction between the RRM of human eIF3b (green and light blue) and the N-terminal peptide of human eIF3j (yellow) (12), the NMR structure of the C-terminal RRM of human eIF3g (red and sky-blue) (5), and the X-ray structure of the yeast i/TIF34–b/PRT1 complex (this study), were used to replace the original schematic representations of the corresponding molecules.
Mentions: To further test whether the mutations under study impair a direct contact between b/PRT1 and i/TIF34 proteins we introduced three single-Ala-substitution mutations into full length b/PRT1, synthesized and radiolabeled the resulting mutant proteins in vitro and tested their binding affinities towards i/TIF34 and g/TIF35 fused with the GST moiety. As shown in Figure 4A, all three completely eliminate binding of b/PRT1 specifically to i/TIF34 but not to g/TIF35. Similarly, both single D/K substitutions and the DD/KK double mutation of i/TIF34, the latter of which was chosen for further analysis, abolished binding of [35S]-labeled i/TIF34 to GST-b/PRT1 but not to GST-g/TIF35 (Figure 4B and data not shown).Figure 4.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus