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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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A model of two eIF3 modules bound to the opposite termini of the scaffold b/PRT1 subunit situated near the mRNA entry channel of the 40S subunit. (Upper panel) The Cryo-EM reconstruction of the 40S subunit is shown from the solvent side with ribosomal RNA represented as tubes. Ribosomal proteins, with known bacterial homologs and placement, are shown as pink cartoons and labeled (adapted from (57)). Positions of RPS0, 2, 3 and 20 and 18S rRNA helices 16–18 are highlighted in bold. The mRNA entry channel is designated by an asterisk. (Lower panel) Hypothetical location of S. cerevisiae eIF3 on the back side of the 40S subunit based on the data presented in this study and elsewhere, including the interactions between RPS0 and a/TIF32-NTD; RPS2 and j/HCR1; RPS2 and 3 and a/TIF32-CTD; helices 16-18 of 18S rRNA and a/TIF32-CTD; and RPS3 and 20 and g/TIF35 (see text for details). The schematic representations of b/PRT1-CTD and i/TIF34 were replaced with the X-ray structure as in Figure 4C. Two eIF3 modules represented by the b/PRT1-CTD–i/TIF34–g/TIF35 and the b/PRT1-RRM–a/TIF32-CTD–j/HCR1 are color-coded in green and blue, respectively. The yellow lines represent mRNA.
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gkr765-F9: A model of two eIF3 modules bound to the opposite termini of the scaffold b/PRT1 subunit situated near the mRNA entry channel of the 40S subunit. (Upper panel) The Cryo-EM reconstruction of the 40S subunit is shown from the solvent side with ribosomal RNA represented as tubes. Ribosomal proteins, with known bacterial homologs and placement, are shown as pink cartoons and labeled (adapted from (57)). Positions of RPS0, 2, 3 and 20 and 18S rRNA helices 16–18 are highlighted in bold. The mRNA entry channel is designated by an asterisk. (Lower panel) Hypothetical location of S. cerevisiae eIF3 on the back side of the 40S subunit based on the data presented in this study and elsewhere, including the interactions between RPS0 and a/TIF32-NTD; RPS2 and j/HCR1; RPS2 and 3 and a/TIF32-CTD; helices 16-18 of 18S rRNA and a/TIF32-CTD; and RPS3 and 20 and g/TIF35 (see text for details). The schematic representations of b/PRT1-CTD and i/TIF34 were replaced with the X-ray structure as in Figure 4C. Two eIF3 modules represented by the b/PRT1-CTD–i/TIF34–g/TIF35 and the b/PRT1-RRM–a/TIF32-CTD–j/HCR1 are color-coded in green and blue, respectively. The yellow lines represent mRNA.

Mentions: Several important intermolecular bridges between yeast eIF3 and the solvent-exposed side of the 40S ribosome were previously identified, including those between the NTD of a/TIF32 and the small ribosomal protein RPS0A, the a/TIF32-CTD and helices 16–18 of 18S rRNA and RPS2 and RPS3, and the CTD of j/HCR1 and RPS2 (12,37,44,48). Note that both RPS2 and 3 are situated near the mRNA entry pore (Figure 9, upper panel). Besides them, the CTD of c/NIP1 and the b/PRT1-RRM were also shown to critically contribute to the eIF3 affinity for the 40S subunit; however, their binding partners remain to be identified (12,37). Whether or not the remaining eIF3 subunits in i/TIF34 and g/TIF35 likewise participate in this functionally crucial eIF3-ribosome binding activity remained unclear until now. On the one hand a partial subcomplex composed of i/TIF34, g/TIF35 and b/PRT1 lacking its N-terminal RRM showed zero 40S-binding affinity in vivo (9). On the other hand, another subcomplex comprising c/NIP1, the critical N-terminal half of a/TIF32, and eIF5 showed a substantial affinity for the 40S subunits in vivo, though not as strong as that of the wt 6-subunit eIF3 (37). Based on these findings and the data presented here, we propose that whereas the major and essential driving force of the 40S-binding affinity of yeast eIF3 lies in the three largest subunits, as proposed earlier (37), i/TIF34 and g/TIF35 provide complementary 40S-binding activity that is required for stabilization of the entire 48S PICs. It is therefore conceivable that the proper establishment of all intermolecular bridges between eIF3 and the 40S ribosome is needed to ensure precise positioning of eIF3 on the small subunit and thereby flawless functioning of eIF3 not only in formation of 43S and 48S PICs, but also in the subsequent initiation steps.Figure 9.


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)

A model of two eIF3 modules bound to the opposite termini of the scaffold b/PRT1 subunit situated near the mRNA entry channel of the 40S subunit. (Upper panel) The Cryo-EM reconstruction of the 40S subunit is shown from the solvent side with ribosomal RNA represented as tubes. Ribosomal proteins, with known bacterial homologs and placement, are shown as pink cartoons and labeled (adapted from (57)). Positions of RPS0, 2, 3 and 20 and 18S rRNA helices 16–18 are highlighted in bold. The mRNA entry channel is designated by an asterisk. (Lower panel) Hypothetical location of S. cerevisiae eIF3 on the back side of the 40S subunit based on the data presented in this study and elsewhere, including the interactions between RPS0 and a/TIF32-NTD; RPS2 and j/HCR1; RPS2 and 3 and a/TIF32-CTD; helices 16-18 of 18S rRNA and a/TIF32-CTD; and RPS3 and 20 and g/TIF35 (see text for details). The schematic representations of b/PRT1-CTD and i/TIF34 were replaced with the X-ray structure as in Figure 4C. Two eIF3 modules represented by the b/PRT1-CTD–i/TIF34–g/TIF35 and the b/PRT1-RRM–a/TIF32-CTD–j/HCR1 are color-coded in green and blue, respectively. The yellow lines represent mRNA.
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Related In: Results  -  Collection

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gkr765-F9: A model of two eIF3 modules bound to the opposite termini of the scaffold b/PRT1 subunit situated near the mRNA entry channel of the 40S subunit. (Upper panel) The Cryo-EM reconstruction of the 40S subunit is shown from the solvent side with ribosomal RNA represented as tubes. Ribosomal proteins, with known bacterial homologs and placement, are shown as pink cartoons and labeled (adapted from (57)). Positions of RPS0, 2, 3 and 20 and 18S rRNA helices 16–18 are highlighted in bold. The mRNA entry channel is designated by an asterisk. (Lower panel) Hypothetical location of S. cerevisiae eIF3 on the back side of the 40S subunit based on the data presented in this study and elsewhere, including the interactions between RPS0 and a/TIF32-NTD; RPS2 and j/HCR1; RPS2 and 3 and a/TIF32-CTD; helices 16-18 of 18S rRNA and a/TIF32-CTD; and RPS3 and 20 and g/TIF35 (see text for details). The schematic representations of b/PRT1-CTD and i/TIF34 were replaced with the X-ray structure as in Figure 4C. Two eIF3 modules represented by the b/PRT1-CTD–i/TIF34–g/TIF35 and the b/PRT1-RRM–a/TIF32-CTD–j/HCR1 are color-coded in green and blue, respectively. The yellow lines represent mRNA.
Mentions: Several important intermolecular bridges between yeast eIF3 and the solvent-exposed side of the 40S ribosome were previously identified, including those between the NTD of a/TIF32 and the small ribosomal protein RPS0A, the a/TIF32-CTD and helices 16–18 of 18S rRNA and RPS2 and RPS3, and the CTD of j/HCR1 and RPS2 (12,37,44,48). Note that both RPS2 and 3 are situated near the mRNA entry pore (Figure 9, upper panel). Besides them, the CTD of c/NIP1 and the b/PRT1-RRM were also shown to critically contribute to the eIF3 affinity for the 40S subunit; however, their binding partners remain to be identified (12,37). Whether or not the remaining eIF3 subunits in i/TIF34 and g/TIF35 likewise participate in this functionally crucial eIF3-ribosome binding activity remained unclear until now. On the one hand a partial subcomplex composed of i/TIF34, g/TIF35 and b/PRT1 lacking its N-terminal RRM showed zero 40S-binding affinity in vivo (9). On the other hand, another subcomplex comprising c/NIP1, the critical N-terminal half of a/TIF32, and eIF5 showed a substantial affinity for the 40S subunits in vivo, though not as strong as that of the wt 6-subunit eIF3 (37). Based on these findings and the data presented here, we propose that whereas the major and essential driving force of the 40S-binding affinity of yeast eIF3 lies in the three largest subunits, as proposed earlier (37), i/TIF34 and g/TIF35 provide complementary 40S-binding activity that is required for stabilization of the entire 48S PICs. It is therefore conceivable that the proper establishment of all intermolecular bridges between eIF3 and the 40S ribosome is needed to ensure precise positioning of eIF3 on the small subunit and thereby flawless functioning of eIF3 not only in formation of 43S and 48S PICs, but also in the subsequent initiation steps.Figure 9.

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