Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis

Abstract

During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon–anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to Pi release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and Pi release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining.

Four translation initiation pathways employed by the leaderless mRNA in eukaryotes

“…By contrast, eIF5 gave a relative advantage to the leaderless mRNA (Fig. 3b), probably by stimulating the 5′-terminal AUG recognition and blocking ribosomal sliding4344. Interestingly, eIF5B, the eukaryotic homolog of bacterial IF2, also differentially affected the reporter mRNAs translation (Fig.…”

“…3b). This could be explained either by stabilization of the 48 S complex and inhibition of its sliding4445 or by stimulation of an alternative translation initiation pathway in a manner similar to bacterial, archaeal, and mitochondrial IF246; see below.…”

“…eIF2, eIF3, eIF4F, eIF5B, 40 S and 60 S were purified from HeLa cell extract, eIF1, eIF1A, eIF4A, eIF4B and eIF5 were expressed in E. coli as described234461. Purified tRNA f Met , a kind gift from V. Makhno and Y. Semenkov, was used as initiator tRNA.…”

Decoding sORF translation – from small proteins to gene regulation

“…108,109 On the other hand, long uORFs and short distances between uORF and main ORF impede reinitiation and thus correlate with increased repressiveness of the uORF. 91,110,111 Regulating the regulator -how uORF translation can be controlled Apart from such a constitutive mode of action, there is increasing evidence that uORF-mediated regulation itself can be dynamically regulated. In its simplest form of regulation, uORFs can either be selectively included or excluded during the production of the mRNA.…”

“…Moreover, changing the activity and concentration of eIF2 affects the general ability of ribosomes to reinitiate translation after uORFs. 91,110,111 Similarly, a study carried out in Arabidopsis thaliana has identified the target-of-rapamycin (TOR) pathway in conjunction with the S6 kinase (S6K) and the plant reinitiation factor eIF3h as more general regulators of reinitiation. 130 Induction of the TOR pathway by the plant hormone auxin leads to S6K-mediated phosphorylation of eIF3h, which promotes the assembly of reinitiating ribosomes.…”