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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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Structure of i/TIF34–b/PRT1 complex. (A) Schematic drawing of the predicted protein domains of yeast i/TIF34 (top) and b/PRT1 subunits of eIF3 (bottom). The folded domains and their boundaries are indicated. Predicted unstructured regions are shown as lines. (B) Overview of the structure: cartoon representation of i/TIF34 (green) and b/PRT1(654–700) (blue). WD-40 blades 1–7, β-strands a–d of blade 1 and N-terminus (nt) and C-terminus (ct) are labeled. The complex is shown from the bottom side of the β-propeller, where loops occur between strands a–b and c–d. (C) Topology diagram of the i/TIF34 fold. (D) Space-filling view of the i/TIF34 in complex with b/PRT1 (bottom view, left and top view, right), colored according to sequence conservation. A gradient of green to purple indicates the degree of phylogenetic conservation, with variable shown as green and most conserved as dark purple. The conservation heat plot of the i/TIF34 surface was generated by ConSurf using multiple alignment of Human, Drosophila, Arabidopsis and Saccharomyces cerevisiae i/TIF34 protein homologues. All structural figures were generated using PyMOL (http://www.pymol.org).
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gkr765-F1: Structure of i/TIF34–b/PRT1 complex. (A) Schematic drawing of the predicted protein domains of yeast i/TIF34 (top) and b/PRT1 subunits of eIF3 (bottom). The folded domains and their boundaries are indicated. Predicted unstructured regions are shown as lines. (B) Overview of the structure: cartoon representation of i/TIF34 (green) and b/PRT1(654–700) (blue). WD-40 blades 1–7, β-strands a–d of blade 1 and N-terminus (nt) and C-terminus (ct) are labeled. The complex is shown from the bottom side of the β-propeller, where loops occur between strands a–b and c–d. (C) Topology diagram of the i/TIF34 fold. (D) Space-filling view of the i/TIF34 in complex with b/PRT1 (bottom view, left and top view, right), colored according to sequence conservation. A gradient of green to purple indicates the degree of phylogenetic conservation, with variable shown as green and most conserved as dark purple. The conservation heat plot of the i/TIF34 surface was generated by ConSurf using multiple alignment of Human, Drosophila, Arabidopsis and Saccharomyces cerevisiae i/TIF34 protein homologues. All structural figures were generated using PyMOL (http://www.pymol.org).

Mentions: Using solution NMR spectroscopy, we defined the minimal C-terminal i/TIF34-binding site of b/PRT1(654–700) lacking any b/PRT1 random coil segments, which could hinder crystallization (16,17) (Figure 1A, Supplementary Figures S1 and S2A, ‘Material and Methods’ section). The crystallographic structure of thus optimized yeast i/TIF34–b/PRT1(654–700) complex was determined at 2.2 Å resolution by MIR heavy atom phasing (data statistics in Supplementary Table S1). Two complexes were found in the asymmetric unit and both of them displayed excellent electron density (Supplementary Figures S3 and S4) except for a few terminal residues of both the b/PRT1 and i/TIF34 molecules and a specific loop region spanning well-conserved residues 260–272 in one of the two i/TIF34 molecules (discussed in more detail later).Figure 1.


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)

Structure of i/TIF34–b/PRT1 complex. (A) Schematic drawing of the predicted protein domains of yeast i/TIF34 (top) and b/PRT1 subunits of eIF3 (bottom). The folded domains and their boundaries are indicated. Predicted unstructured regions are shown as lines. (B) Overview of the structure: cartoon representation of i/TIF34 (green) and b/PRT1(654–700) (blue). WD-40 blades 1–7, β-strands a–d of blade 1 and N-terminus (nt) and C-terminus (ct) are labeled. The complex is shown from the bottom side of the β-propeller, where loops occur between strands a–b and c–d. (C) Topology diagram of the i/TIF34 fold. (D) Space-filling view of the i/TIF34 in complex with b/PRT1 (bottom view, left and top view, right), colored according to sequence conservation. A gradient of green to purple indicates the degree of phylogenetic conservation, with variable shown as green and most conserved as dark purple. The conservation heat plot of the i/TIF34 surface was generated by ConSurf using multiple alignment of Human, Drosophila, Arabidopsis and Saccharomyces cerevisiae i/TIF34 protein homologues. All structural figures were generated using PyMOL (http://www.pymol.org).
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Related In: Results  -  Collection

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

gkr765-F1: Structure of i/TIF34–b/PRT1 complex. (A) Schematic drawing of the predicted protein domains of yeast i/TIF34 (top) and b/PRT1 subunits of eIF3 (bottom). The folded domains and their boundaries are indicated. Predicted unstructured regions are shown as lines. (B) Overview of the structure: cartoon representation of i/TIF34 (green) and b/PRT1(654–700) (blue). WD-40 blades 1–7, β-strands a–d of blade 1 and N-terminus (nt) and C-terminus (ct) are labeled. The complex is shown from the bottom side of the β-propeller, where loops occur between strands a–b and c–d. (C) Topology diagram of the i/TIF34 fold. (D) Space-filling view of the i/TIF34 in complex with b/PRT1 (bottom view, left and top view, right), colored according to sequence conservation. A gradient of green to purple indicates the degree of phylogenetic conservation, with variable shown as green and most conserved as dark purple. The conservation heat plot of the i/TIF34 surface was generated by ConSurf using multiple alignment of Human, Drosophila, Arabidopsis and Saccharomyces cerevisiae i/TIF34 protein homologues. All structural figures were generated using PyMOL (http://www.pymol.org).
Mentions: Using solution NMR spectroscopy, we defined the minimal C-terminal i/TIF34-binding site of b/PRT1(654–700) lacking any b/PRT1 random coil segments, which could hinder crystallization (16,17) (Figure 1A, Supplementary Figures S1 and S2A, ‘Material and Methods’ section). The crystallographic structure of thus optimized yeast i/TIF34–b/PRT1(654–700) complex was determined at 2.2 Å resolution by MIR heavy atom phasing (data statistics in Supplementary Table S1). Two complexes were found in the asymmetric unit and both of them displayed excellent electron density (Supplementary Figures S3 and S4) except for a few terminal residues of both the b/PRT1 and i/TIF34 molecules and a specific loop region spanning well-conserved residues 260–272 in one of the two i/TIF34 molecules (discussed in more detail later).Figure 1.

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