In Vitro Reconstitution of Eukaryotic Translation Reveals Cooperativity between Release Factors eRF1 and eRF3

Abstract

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a 2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1.

Cryo-EM structure of the mammalian eukaryotic release factor eRF1–eRF3-associated termination complex

“…eRF3 strongly stimulates the process in the presence of GTP, but this stimulation is completely abrogated in the presence of the nonhydrolyzable GTP analog guanosine 5′-[β,γ-imido]triphosphate (GMPPNP) (23). This indicates that eRF3's GTPase activity couples stop codon recognition with hydrolysis of peptidyl-tRNA, as first suggested on the basis of genetic data (24).…”

“…Mammalian pre-TCs were obtained by in vitro reconstitution from purified rabbit 40S and 60S ribosomal subunits, initiation (2, 3, 1, 1A, 4A, 4B, 4G, 5, and 5B), and elongation factors (1A and 2), and aminoacylated tRNAs on MVHL-STOP (Methionine-valine-histidine-leucine-stop codon) mRNA comprising 12 unstructured 5′-terminal nucleotides (four CAA triplets) followed by the β-globin 5′-UTR, a coding sequence for the MVHL tetrapeptide, a UAA stop codon, and an ∼150-nt 3′-UTR consisting of the natural β-globin coding sequence (23). Assembled pre-TCs were purified by sucrose density gradient (SDG) centrifugation.…”

“…eRF1-eRF3-associated ribosomal complexes, corresponding to the pre-GTP hydrolysis stage of initial attachment of release factors, were obtained by incubation of SDGpurified pre-TCs formed on MVHL-STOP mRNA with the fulllength eRF1 and truncated eRF3 lacking the nonessential 138 N-terminal amino acids (21,23) in the presence of GMPPNP. The efficiency of pretermination complex formation, estimated by incorporation of [ 35 S]Met, was ∼70%.…”

“…In such a conformation, domain M would come in direct contact with the switch regions of the eRF3 G domain. Interestingly, binding of eRF1-eRF3-GTP to ribosomal pretermination complexes (preTCs) also induces a +2-nt forward shift in their mRNA toeprints (23), which could be due to additional conformational changes in the 80S ribosome.…”

Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation

“…The activation of eRF3 GTPase activity strictly requires the presence of both eRF1 and the ribosome (Frolova et al 1996). Recent data suggest that GTP hydrolysis by eRF3 couples stop codon recognition by eRF1 with peptidyl-tRNA hydrolysis by the peptidyl transferase center (Salas-Marco and Bedwell 2004; Alkalaeva et al 2006). eRF1 and eRF3 form a stable complex through interaction of their C-terminal domains (Ito et al 1998;Ebihara and Nakamura 1999;Merkulova et al 1999).…”