To facilitate translation elongation the GTPase EF-Tu in bacteria (eEF1A in eukaryotes) forms a ternary complex (TC) with GTP and an aminoacyl-tRNA, and then binds the aminoacyl-tRNA to the A-site of the ribosome in a codon-dependent manner. Recent x-ray crystallographic studies have provided a high resolution image of the EF-Tu–GTP–aminoacyl-tRNA–70S ribosome complex and revealed contacts between EF-Tu and rRNA elements near the A-site of the ribosome1,2
. Functioning in an analogous manner to EF-Tu, the eukaryote-specific translation initiation factor eIF2 forms a TC with GTP and the specific initiator Met-tRNAiMet
. However, in contrast to EF-Tu, which interacts with 70S elongating ribosomes, eIF2 binds Met-tRNAiMet
to the small (40S) ribosomal subunit in eukaryotic cells to help form the 43S preinitiation complex (PIC) (reviewed in3
). Binding of the factors eIF1 and eIF1A to the 40S subunit enhances TC binding, and creates an open, scanning-competent complex4–6
. The resulting PIC binds an mRNA near the 5′ cap and then this 48S PIC scans the mRNA in search of a start codon. Base-pairing between the anticodon of the Met-tRNAiMet
and a start codon in the mRNA triggers reconfiguration of the PIC from the open to a closed, scanning-arrested state. This open to closed transition is accompanied by movement of eIF1 away from eIF1A, completion of GTP hydrolysis, and release of Pi7
. Joining of the 60S subunit yields an 80S ribosome with Met-tRNAiMet
in the P-site base-paired with the start codon on the mRNA.
Previous x-ray crystallography and structural probing studies have provided insights into the ribosomal binding sites of several of the PIC factors. Directed hydroxyl radical mapping using factor tethered Fe(II) placed the core of eIF1A in the A-site of the 40S subunit8
. This location is consistent with the x-ray structure of the analogous bacterial initiation complex which showed IF1 binding to the A-site of the 30S subunit9
. Interestingly, the N-terminal and C-terminal tails of eIF1A extend from the A-site toward and under the P-site bound Met-tRNAiMet
). Whereas the N-terminal tail does not contact the P-site, the C-terminal tail of eIF1A contacts the P-site and is proposed to interfere with Met-tRNAiMet
fully entering the P-site (Pout
state) in the open scanning complex8,10
. Meanwhile, eIF1, which plays a critical role in start codon selection, binds near the P-site in a location where it might monitor codon-anticodon interactions that govern start site selection5
. Upon start codon recognition and conversion of the PIC to the closed scanning arrested state, Met-tRNAiMet
fully docks in the P-site (Pin
state) displacing both the eIF1A C-terminal tail and eIF18,10
. While eIF2 is known to bind Met-tRNAiMet
, which is bound to the P-site of PICs, the binding site of eIF2 on the ribosome has not been determined.
The γ subunit of the heterotrimeric eIF2 complex shares significant amino acid sequence and structural similarity with EF-Tu (, and Supplementary Fig. 1 online
). Whereas no structures have been obtained for eIF2γ, x-ray crystal structures have been obtained for the corresponding archaeal aIF2γ protein both free11,12
and in complex with full-length or truncated versions of aIF2α and aIF2β13–15,16
(). Both aIF2γ and EF-Tu consist of three domains: an N-terminal GTP-binding domain, and β-barrel domains II and III (, and Supplementary Fig. 1 online
). Despite the structural similarity, it is anticipated that eIF2γ and EF-Tu will have different docking arrangements on the A-versus P-sites of the ribosome, and this might lead to differences in how the two factors bind aminoacyl-tRNAs. Moreover, despite the wealth of structural information on aIF2 complexes, the ribosome-contacting surfaces aIF2 and eIF2 have not been identified. To gain insights into how eIF2 binds Met-tRNAiMet
and then associates with the ribosome, we used directed hydroxyl radical probing to identify eIF2 contacts within the 48S PIC.
Structures of EF-Tu TC and aIF2γ TC model.
Construction and analysis of eIF2 Cys mutants.