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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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Molecular details of i/TIF34 and b/PRT1 interactions. (A) Electrostatic potential (±5kT/e) of the solvent-accessible surface of i/TIF34 in complex with b/PRT1(654–700) rendered on the molecular surface of the complex. A gradient of blue to red shows positive to negative charge, respectively, as calculated using PyMOL built-in APBS tools (55). PQR file for analysis of Poisson–Boltzmann electrostatics calculations was generated using PDB2PQR tool (56) and further used for APBS. b/PRT1(654–700) is shown as a cartoon in blue. View from the b/PRT1 binding site is shown on the left; the ‘reverse’ side of i/TIF34 shown on the right has negative charge clustering at the highly conserved blade 1 (upper part), blades 4 and 5 (the bottom part) and positive charge around the central cavity. (B–C) The i/TIF34-b/PRT1 binding interface involves highly conserved amino acids from both proteins. i/TIF34 is shown in green, b/PRT1 in blue. (B) Y677 and R678 from b/PRT1 form H-bonds (blue) with D207, T209 and D224 from i/TIF34 (only interacting side chains are shown). Other residues making contacts (light blue) via main chain atoms are also shown in sticks. One water molecule (shown in dots) is in close proximity (light pink) to R678 NH1 (2.6 Å) and Y677 OH (3.3 Å). (C) b/PRT1 W674 is surrounded by hydrophobic and charged amino acids (only side chains are shown), which form a shallow pocket. (D) Surface representation of the i/TIF34 hydrophobic pocket, which accommodates b/PRT1 W674. This interaction serves as a ‘lock’ for the i/TIF34 and b/PRT1 interaction interface.
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gkr765-F2: Molecular details of i/TIF34 and b/PRT1 interactions. (A) Electrostatic potential (±5kT/e) of the solvent-accessible surface of i/TIF34 in complex with b/PRT1(654–700) rendered on the molecular surface of the complex. A gradient of blue to red shows positive to negative charge, respectively, as calculated using PyMOL built-in APBS tools (55). PQR file for analysis of Poisson–Boltzmann electrostatics calculations was generated using PDB2PQR tool (56) and further used for APBS. b/PRT1(654–700) is shown as a cartoon in blue. View from the b/PRT1 binding site is shown on the left; the ‘reverse’ side of i/TIF34 shown on the right has negative charge clustering at the highly conserved blade 1 (upper part), blades 4 and 5 (the bottom part) and positive charge around the central cavity. (B–C) The i/TIF34-b/PRT1 binding interface involves highly conserved amino acids from both proteins. i/TIF34 is shown in green, b/PRT1 in blue. (B) Y677 and R678 from b/PRT1 form H-bonds (blue) with D207, T209 and D224 from i/TIF34 (only interacting side chains are shown). Other residues making contacts (light blue) via main chain atoms are also shown in sticks. One water molecule (shown in dots) is in close proximity (light pink) to R678 NH1 (2.6 Å) and Y677 OH (3.3 Å). (C) b/PRT1 W674 is surrounded by hydrophobic and charged amino acids (only side chains are shown), which form a shallow pocket. (D) Surface representation of the i/TIF34 hydrophobic pocket, which accommodates b/PRT1 W674. This interaction serves as a ‘lock’ for the i/TIF34 and b/PRT1 interaction interface.

Mentions: The b/PRT1-binding interface of i/TIF34 comprises residues from β-sheets and loops in blades 5 and 6 (Figure 1B). The total buried surface area of the b/PRT1–i/TIF34 interface is 1028.7 Å2 and 1054.6 Å2 (complex I and complex II, respectively, in Supplementary Figure S4) as calculated using PISA (35). The b/PRT1-interacting surface of i/TIF34 bears a few important charged regions (Figure 2A; left). The remaining surface of this side of the β-propeller shows dominantly negative charge, while patches of positive charge dominate around the central cavity on the top side of the β-propeller (formed by blades 2–5, Figure 2A; right). This charge distribution is conserved suggesting distinct interfaces for the interactions with other components of the translational machinery (Figures 1D, 2A and Supplementary Figure S2A).Figure 2.


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)

Molecular details of i/TIF34 and b/PRT1 interactions. (A) Electrostatic potential (±5kT/e) of the solvent-accessible surface of i/TIF34 in complex with b/PRT1(654–700) rendered on the molecular surface of the complex. A gradient of blue to red shows positive to negative charge, respectively, as calculated using PyMOL built-in APBS tools (55). PQR file for analysis of Poisson–Boltzmann electrostatics calculations was generated using PDB2PQR tool (56) and further used for APBS. b/PRT1(654–700) is shown as a cartoon in blue. View from the b/PRT1 binding site is shown on the left; the ‘reverse’ side of i/TIF34 shown on the right has negative charge clustering at the highly conserved blade 1 (upper part), blades 4 and 5 (the bottom part) and positive charge around the central cavity. (B–C) The i/TIF34-b/PRT1 binding interface involves highly conserved amino acids from both proteins. i/TIF34 is shown in green, b/PRT1 in blue. (B) Y677 and R678 from b/PRT1 form H-bonds (blue) with D207, T209 and D224 from i/TIF34 (only interacting side chains are shown). Other residues making contacts (light blue) via main chain atoms are also shown in sticks. One water molecule (shown in dots) is in close proximity (light pink) to R678 NH1 (2.6 Å) and Y677 OH (3.3 Å). (C) b/PRT1 W674 is surrounded by hydrophobic and charged amino acids (only side chains are shown), which form a shallow pocket. (D) Surface representation of the i/TIF34 hydrophobic pocket, which accommodates b/PRT1 W674. This interaction serves as a ‘lock’ for the i/TIF34 and b/PRT1 interaction interface.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr765-F2: Molecular details of i/TIF34 and b/PRT1 interactions. (A) Electrostatic potential (±5kT/e) of the solvent-accessible surface of i/TIF34 in complex with b/PRT1(654–700) rendered on the molecular surface of the complex. A gradient of blue to red shows positive to negative charge, respectively, as calculated using PyMOL built-in APBS tools (55). PQR file for analysis of Poisson–Boltzmann electrostatics calculations was generated using PDB2PQR tool (56) and further used for APBS. b/PRT1(654–700) is shown as a cartoon in blue. View from the b/PRT1 binding site is shown on the left; the ‘reverse’ side of i/TIF34 shown on the right has negative charge clustering at the highly conserved blade 1 (upper part), blades 4 and 5 (the bottom part) and positive charge around the central cavity. (B–C) The i/TIF34-b/PRT1 binding interface involves highly conserved amino acids from both proteins. i/TIF34 is shown in green, b/PRT1 in blue. (B) Y677 and R678 from b/PRT1 form H-bonds (blue) with D207, T209 and D224 from i/TIF34 (only interacting side chains are shown). Other residues making contacts (light blue) via main chain atoms are also shown in sticks. One water molecule (shown in dots) is in close proximity (light pink) to R678 NH1 (2.6 Å) and Y677 OH (3.3 Å). (C) b/PRT1 W674 is surrounded by hydrophobic and charged amino acids (only side chains are shown), which form a shallow pocket. (D) Surface representation of the i/TIF34 hydrophobic pocket, which accommodates b/PRT1 W674. This interaction serves as a ‘lock’ for the i/TIF34 and b/PRT1 interaction interface.
Mentions: The b/PRT1-binding interface of i/TIF34 comprises residues from β-sheets and loops in blades 5 and 6 (Figure 1B). The total buried surface area of the b/PRT1–i/TIF34 interface is 1028.7 Å2 and 1054.6 Å2 (complex I and complex II, respectively, in Supplementary Figure S4) as calculated using PISA (35). The b/PRT1-interacting surface of i/TIF34 bears a few important charged regions (Figure 2A; left). The remaining surface of this side of the β-propeller shows dominantly negative charge, while patches of positive charge dominate around the central cavity on the top side of the β-propeller (formed by blades 2–5, Figure 2A; right). This charge distribution is conserved suggesting distinct interfaces for the interactions with other components of the translational machinery (Figures 1D, 2A and Supplementary Figure S2A).Figure 2.

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