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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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Phenotypic and biochemical analysis of i/TIF34 and b/PRT1 mutations that disrupt subunit interactions. (A) The prt1-W674A and -YR/AA but not -W674F mutations produce severe slow growth and temperature sensitive phenotypes. The YAH06 (prt1Δ) strain was transformed with the corresponding plasmids carrying individual mutant alleles and the resident pCR52 (PRT1,URA3) covering plasmid was evicted on 5-FOA. The resulting strains were then spotted in four serial 10-fold dilutions on SD medium and incubated at 30, 34 and 37°C. (B) The tif34-DD/KK, -L222D and L222K mutations produce severe slow growth and temperature sensitive phenotypes. The H450 (tif34Δ) strain was transformed with the corresponding plasmids carrying individual mutant alleles and the resident YEp-i/TIF34-U (TIF34, URA3) covering plasmid was evicted on 5-FOA. The resulting strains were then spotted in four serial 10-fold dilutions on SD medium and incubated at 30, 34 and 37°C. (C) Summary of phenotypes of mutations analyzed in this study.
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gkr765-F3: Phenotypic and biochemical analysis of i/TIF34 and b/PRT1 mutations that disrupt subunit interactions. (A) The prt1-W674A and -YR/AA but not -W674F mutations produce severe slow growth and temperature sensitive phenotypes. The YAH06 (prt1Δ) strain was transformed with the corresponding plasmids carrying individual mutant alleles and the resident pCR52 (PRT1,URA3) covering plasmid was evicted on 5-FOA. The resulting strains were then spotted in four serial 10-fold dilutions on SD medium and incubated at 30, 34 and 37°C. (B) The tif34-DD/KK, -L222D and L222K mutations produce severe slow growth and temperature sensitive phenotypes. The H450 (tif34Δ) strain was transformed with the corresponding plasmids carrying individual mutant alleles and the resident YEp-i/TIF34-U (TIF34, URA3) covering plasmid was evicted on 5-FOA. The resulting strains were then spotted in four serial 10-fold dilutions on SD medium and incubated at 30, 34 and 37°C. (C) Summary of phenotypes of mutations analyzed in this study.

Mentions: To confirm the critical aspects of the b/PRT1–i/TIF34 contacts revealed by the structure analysis and to investigate the functional consequences of their loss in living cells, we first substituted Y677 and R678 (alone or in combination), or W674 of b/PRT1 with Ala, Asp or Phe residues, respectively, and tested them for growth phenotypes. The corresponding mutations were generated in a plasmid copy of PRT1-His, encoding His8-tagged b/PRT1, and introduced into a prt1Δ strain harboring wt PRT1 on a URA3 plasmid, which was subsequently evicted by counter-selection on medium containing 5-fluoroorotic acid (5-FOA). Whereas the double Y677A R678D mutation is lethal and the Y677A R678A (dubbed YR/AA herein) double mutant imparts a very severe slow growth (Slg–) phenotype, individual Y677A and R678A substitutions show little to no effect on growth rates (Figure 3A). Severe Slg− and temperature sensitive (Ts−) phenotypes are also found associated with the W674A mutation. Strikingly, the ‘phylogenetic correction’ substitution in W674F (see above) displays wt-like behavior under all tested conditions (Figure 3A; summarized in 3C). Importantly, in vitro electrophoretic mobility shift assays performed with recombinant purified i/TIF34 and b/PRT1-CTD(630–724) variants confirm these in vivo results: b/prt1-W674F mutant protein competes well with wt b/PRT1-CTD for i/TIF34 binding, whereas b/prt1-W674A and the YR/AA double mutant are not able to compete for i/TIF34 binding at all (Supplementary Figure S7). These results confirm that an aromatic side chain (Phe or Trp) is required to fill the hydrophobic pocket on i/TIF34 and indicate that the contacts of Y677 and R678 with D207 and D224, respectively, are redundant (see more biochemical evidence further below).Figure 3.


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)

Phenotypic and biochemical analysis of i/TIF34 and b/PRT1 mutations that disrupt subunit interactions. (A) The prt1-W674A and -YR/AA but not -W674F mutations produce severe slow growth and temperature sensitive phenotypes. The YAH06 (prt1Δ) strain was transformed with the corresponding plasmids carrying individual mutant alleles and the resident pCR52 (PRT1,URA3) covering plasmid was evicted on 5-FOA. The resulting strains were then spotted in four serial 10-fold dilutions on SD medium and incubated at 30, 34 and 37°C. (B) The tif34-DD/KK, -L222D and L222K mutations produce severe slow growth and temperature sensitive phenotypes. The H450 (tif34Δ) strain was transformed with the corresponding plasmids carrying individual mutant alleles and the resident YEp-i/TIF34-U (TIF34, URA3) covering plasmid was evicted on 5-FOA. The resulting strains were then spotted in four serial 10-fold dilutions on SD medium and incubated at 30, 34 and 37°C. (C) Summary of phenotypes of mutations analyzed in this study.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3300007&req=5

gkr765-F3: Phenotypic and biochemical analysis of i/TIF34 and b/PRT1 mutations that disrupt subunit interactions. (A) The prt1-W674A and -YR/AA but not -W674F mutations produce severe slow growth and temperature sensitive phenotypes. The YAH06 (prt1Δ) strain was transformed with the corresponding plasmids carrying individual mutant alleles and the resident pCR52 (PRT1,URA3) covering plasmid was evicted on 5-FOA. The resulting strains were then spotted in four serial 10-fold dilutions on SD medium and incubated at 30, 34 and 37°C. (B) The tif34-DD/KK, -L222D and L222K mutations produce severe slow growth and temperature sensitive phenotypes. The H450 (tif34Δ) strain was transformed with the corresponding plasmids carrying individual mutant alleles and the resident YEp-i/TIF34-U (TIF34, URA3) covering plasmid was evicted on 5-FOA. The resulting strains were then spotted in four serial 10-fold dilutions on SD medium and incubated at 30, 34 and 37°C. (C) Summary of phenotypes of mutations analyzed in this study.
Mentions: To confirm the critical aspects of the b/PRT1–i/TIF34 contacts revealed by the structure analysis and to investigate the functional consequences of their loss in living cells, we first substituted Y677 and R678 (alone or in combination), or W674 of b/PRT1 with Ala, Asp or Phe residues, respectively, and tested them for growth phenotypes. The corresponding mutations were generated in a plasmid copy of PRT1-His, encoding His8-tagged b/PRT1, and introduced into a prt1Δ strain harboring wt PRT1 on a URA3 plasmid, which was subsequently evicted by counter-selection on medium containing 5-fluoroorotic acid (5-FOA). Whereas the double Y677A R678D mutation is lethal and the Y677A R678A (dubbed YR/AA herein) double mutant imparts a very severe slow growth (Slg–) phenotype, individual Y677A and R678A substitutions show little to no effect on growth rates (Figure 3A). Severe Slg− and temperature sensitive (Ts−) phenotypes are also found associated with the W674A mutation. Strikingly, the ‘phylogenetic correction’ substitution in W674F (see above) displays wt-like behavior under all tested conditions (Figure 3A; summarized in 3C). Importantly, in vitro electrophoretic mobility shift assays performed with recombinant purified i/TIF34 and b/PRT1-CTD(630–724) variants confirm these in vivo results: b/prt1-W674F mutant protein competes well with wt b/PRT1-CTD for i/TIF34 binding, whereas b/prt1-W674A and the YR/AA double mutant are not able to compete for i/TIF34 binding at all (Supplementary Figure S7). These results confirm that an aromatic side chain (Phe or Trp) is required to fill the hydrophobic pocket on i/TIF34 and indicate that the contacts of Y677 and R678 with D207 and D224, respectively, are redundant (see more biochemical evidence further below).Figure 3.

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