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Structural changes enable start codon recognition by the eukaryotic translation initiation complex.

Hussain T, Llácer JL, Fernández IS, Munoz A, Martin-Marcos P, Savva CG, Lorsch JR, Hinnebusch AG, Ramakrishnan V - Cell (2014)

Bottom Line: During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit.The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2.The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

ABSTRACT
During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

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Cryo-EM Structure of the Eukaryotic Preinitiation Complex in the PIN State(A and B) CryoEM maps of the py48S PIC at 4.0 Å shown in two orientations. Regions of the map are colored by component to show the 40S subunit (yellow), eIF1A (blue), eIF1 (cyan), Met-tRNAiMet (green), mRNA (magenta), eIF2α (violet), eIF2γ (orange), and eIF2β (red). Density which may correspond to eIF5 is shown in pink. The density for eIF2β, eIF2γ, and eIF5 is low-pass filtered to 8 Å. The same colors are used in all the figures.(C) Maps at 4.0 Å for tRNAi, mRNA, and eIF2α contoured at 3σ and eIF1 contoured at 2σ.(D) Atomic model for the PIC in the same colors except that 40S proteins and rRNA are displayed in various shades of yellow. rRNA helix 44 is displayed brown.See also Figures S1–S4 and Table S1.
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fig1: Cryo-EM Structure of the Eukaryotic Preinitiation Complex in the PIN State(A and B) CryoEM maps of the py48S PIC at 4.0 Å shown in two orientations. Regions of the map are colored by component to show the 40S subunit (yellow), eIF1A (blue), eIF1 (cyan), Met-tRNAiMet (green), mRNA (magenta), eIF2α (violet), eIF2γ (orange), and eIF2β (red). Density which may correspond to eIF5 is shown in pink. The density for eIF2β, eIF2γ, and eIF5 is low-pass filtered to 8 Å. The same colors are used in all the figures.(C) Maps at 4.0 Å for tRNAi, mRNA, and eIF2α contoured at 3σ and eIF1 contoured at 2σ.(D) Atomic model for the PIC in the same colors except that 40S proteins and rRNA are displayed in various shades of yellow. rRNA helix 44 is displayed brown.See also Figures S1–S4 and Table S1.

Mentions: The structure of the py48S was determined to an overall resolution of 4.0 Å by single-particle electron cryomicroscopy (cryoEM; Figures 1 and S2 and Table S1). The local resolution and the corresponding density were best in the core of the 40S subunit and for components directly attached to it (Figure S3), where it was possible to see side chains for amino acids (Figure S3C). There is clear density for 40S, eIF1, eIF1A, mRNA, tRNAi, and eIF2α (Figures 1A–1C), which allowed the structures of these components to be modeled and refined. Factors eIF1A and eIF1 are respectively observed in the A site and adjacent to the P site, consistent with previous crystal structures of 40S PICs (Rabl et al., 2011; Weisser et al., 2013; Lomakin and Steitz, 2013). The entire mRNA is visible in the cleft in the 40S subunit and presents the start codon in the P site, where it interacts with the tRNAi. eIF2α is bound in the E site alongside the tRNAi, having a large interface with it. Further away from the 40S platform, eIF2γ is attached to the 3′ end of the tRNAi acceptor arm (Figures 1A and 1B). The resolution is worse with distance from the 40S (Figures S3A and S3B) probably due to increased mobility. Therefore, no model building or refinement was done for eIF2γ, and its placement (Figure 1D) was based on the structure of the archaeal TC (Schmitt et al., 2012). There is also no interpretable density for eIF2β and eIF5, although as discussed below, we observed additional density that may originate from parts of these factors (Figure 1B, pink). We do not observe density for eIF3, but its inclusion during complex formation resulted in twice as many particles containing TC, consistent with its ability to enhance PIC formation (Mitchell et al., 2010). It may subsequently have dissociated or become disordered.


Structural changes enable start codon recognition by the eukaryotic translation initiation complex.

Hussain T, Llácer JL, Fernández IS, Munoz A, Martin-Marcos P, Savva CG, Lorsch JR, Hinnebusch AG, Ramakrishnan V - Cell (2014)

Cryo-EM Structure of the Eukaryotic Preinitiation Complex in the PIN State(A and B) CryoEM maps of the py48S PIC at 4.0 Å shown in two orientations. Regions of the map are colored by component to show the 40S subunit (yellow), eIF1A (blue), eIF1 (cyan), Met-tRNAiMet (green), mRNA (magenta), eIF2α (violet), eIF2γ (orange), and eIF2β (red). Density which may correspond to eIF5 is shown in pink. The density for eIF2β, eIF2γ, and eIF5 is low-pass filtered to 8 Å. The same colors are used in all the figures.(C) Maps at 4.0 Å for tRNAi, mRNA, and eIF2α contoured at 3σ and eIF1 contoured at 2σ.(D) Atomic model for the PIC in the same colors except that 40S proteins and rRNA are displayed in various shades of yellow. rRNA helix 44 is displayed brown.See also Figures S1–S4 and Table S1.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4217140&req=5

fig1: Cryo-EM Structure of the Eukaryotic Preinitiation Complex in the PIN State(A and B) CryoEM maps of the py48S PIC at 4.0 Å shown in two orientations. Regions of the map are colored by component to show the 40S subunit (yellow), eIF1A (blue), eIF1 (cyan), Met-tRNAiMet (green), mRNA (magenta), eIF2α (violet), eIF2γ (orange), and eIF2β (red). Density which may correspond to eIF5 is shown in pink. The density for eIF2β, eIF2γ, and eIF5 is low-pass filtered to 8 Å. The same colors are used in all the figures.(C) Maps at 4.0 Å for tRNAi, mRNA, and eIF2α contoured at 3σ and eIF1 contoured at 2σ.(D) Atomic model for the PIC in the same colors except that 40S proteins and rRNA are displayed in various shades of yellow. rRNA helix 44 is displayed brown.See also Figures S1–S4 and Table S1.
Mentions: The structure of the py48S was determined to an overall resolution of 4.0 Å by single-particle electron cryomicroscopy (cryoEM; Figures 1 and S2 and Table S1). The local resolution and the corresponding density were best in the core of the 40S subunit and for components directly attached to it (Figure S3), where it was possible to see side chains for amino acids (Figure S3C). There is clear density for 40S, eIF1, eIF1A, mRNA, tRNAi, and eIF2α (Figures 1A–1C), which allowed the structures of these components to be modeled and refined. Factors eIF1A and eIF1 are respectively observed in the A site and adjacent to the P site, consistent with previous crystal structures of 40S PICs (Rabl et al., 2011; Weisser et al., 2013; Lomakin and Steitz, 2013). The entire mRNA is visible in the cleft in the 40S subunit and presents the start codon in the P site, where it interacts with the tRNAi. eIF2α is bound in the E site alongside the tRNAi, having a large interface with it. Further away from the 40S platform, eIF2γ is attached to the 3′ end of the tRNAi acceptor arm (Figures 1A and 1B). The resolution is worse with distance from the 40S (Figures S3A and S3B) probably due to increased mobility. Therefore, no model building or refinement was done for eIF2γ, and its placement (Figure 1D) was based on the structure of the archaeal TC (Schmitt et al., 2012). There is also no interpretable density for eIF2β and eIF5, although as discussed below, we observed additional density that may originate from parts of these factors (Figure 1B, pink). We do not observe density for eIF3, but its inclusion during complex formation resulted in twice as many particles containing TC, consistent with its ability to enhance PIC formation (Mitchell et al., 2010). It may subsequently have dissociated or become disordered.

Bottom Line: During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit.The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2.The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

ABSTRACT
During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

Show MeSH
Related in: MedlinePlus