Abstract
SummaryDuring 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 eIF2α, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.
Highlights
Eukaryotic translation initiation involves at least 12 initiation factors (Hinnebusch, 2014)
We present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Aresolution with initiator tRNA in the PIN state, prior to eIF1 release
A lower-resolution structure of tRNAi base paired with AUG in a partial mammalian 48S preinitiation complex (PIC) containing eIF1A, mRNA, and deacylated tRNAi but lacking eIF2, eIF1, eIF5, eIF3, or eIF4F (Lomakin and Steitz, 2013; hereafter referred to as pm48S) suggested that a clash between eIF1 and tRNAi bound in the PIN state would be instrumental in disrupting eIF1 interaction with the 40S subunit, leading to eIF1 release from the PIC and subsequent events occurring downstream of AUG recognition
Summary
Eukaryotic translation initiation involves at least 12 initiation factors (eIFs) (Hinnebusch, 2014). Base pairing of tRNAi with an AUG triplet evokes a rearrangement of factors in the PIC—including displacement of eIF1 and possibly the eIF1A CTT from their locations near the P site—and movement of the eIF1A CTT toward the GAP domain of eIF5 (Saini et al, 2010; Yu et al, 2009; Nanda et al, 2013) These rearrangements enable dissociation of eIF1 from the 40S subunit (Maag et al, 2005; Cheung et al, 2007; Martin-Marcos et al, 2013), evoking a closed, scanning-arrested conformation of the 40S subunit and Pi release from eIF2-GDP (Algire et al, 2005). The unstructured N-terminal tail (NTT) of eIF1A promotes isomerization from POUT to PIN, enhancing start codon recognition (Saini et al, 2010), but it is unknown how the NTT functions at the molecular level
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