Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets

Cells reprogram gene expression in response to environmental changes by mobilizing transcriptional activators. The activator protein Gcn4 of the yeast Saccharomyces cerevisiae is regulated by an intricate translational control mechanism, which is the primary focus of this review, and also by the modulation of its stability in response to nutrient availability. Translation of GCN4 mRNA is derepressed in amino acid-deprived cells, leading to transcriptional induction of nearly all genes encoding amino acid biosynthetic enzymes. The trans-acting proteins that control GCN4 translation have general functions in the initiation of protein synthesis, or regulate the activities of initiation factors, so that the molecular events that induce GCN4 translation also reduce the rate of general protein synthesis. This dual regulatory response enables cells to limit their consumption of amino acids while diverting resources into amino acid biosynthesis in nutrient-poor environments. Remarkably, mammalian cells use the same strategy to downregulate protein synthesis while inducing transcriptional activators of stress-response genes under various stressful conditions, including amino acid starvation.

Translational regulation of GCN4 and the general amino acid control of yeast.

Regulation of translation via mRNA structure in prokaryotes and eukaryotes.

Gene-Specific Regulation by General Translation Factors

In eukaryotes, the translation initiation codon is generally identified by the scanning mechanism, wherein every triplet in the messenger RNA leader is inspected for complementarity to the anticodon of methionyl initiator transfer RNA (Met-tRNAi). Binding of Met-tRNAi to the small (40S) ribosomal subunit, in a ternary complex (TC) with eIF2-GTP, is stimulated by eukaryotic initiation factor 1 (eIF1), eIF1A, eIF3, and eIF5, and the resulting preinitiation complex (PIC) joins the 5' end of mRNA preactivated by eIF4F and poly(A)-binding protein. RNA helicases remove secondary structures that impede ribosome attachment and subsequent scanning. Hydrolysis of eIF2-bound GTP is stimulated by eIF5 in the scanning PIC, but completion of the reaction is impeded at non-AUG triplets. Although eIF1 and eIF1A promote scanning, eIF1 and possibly the C-terminal tail of eIF1A must be displaced from the P decoding site to permit base-pairing between Met-tRNAi and the AUG codon, as well as to allow subsequent phosphate release from eIF2-GDP. A second GTPase, eIF5B, catalyzes the joining of the 60S subunit to produce an 80S initiation complex that is competent for elongation.

The Scanning Mechanism of Eukaryotic Translation Initiation

https://www.cell.com/molecular-cell/pdf/S1097-2765(13)00326-2.pdf

eIF5A promotes translation of polyproline motifs.

Initiation factor 3 (eIF3) forms a multifactor complex (MFC) with eIF1, eIF2, and eIF5 that stimulates Met-tRNA(i)(Met) binding to 40S ribosomes and promotes scanning or AUG recognition. We have previously characterized MFC subcomplexes produced in vivo from affinity-tagged eIF3 subunits lacking discrete binding domains for other MFC components. Here we asked whether these subcomplexes can bind to 40S ribosomes in vivo. We found that the N- and C-terminal domains of NIP1/eIF3c, the N- and C-terminal domains of TIF32/eIF3a, and eIF5 have critical functions in 40S binding, with eIF5 and the TIF32-CTD performing redundant functions. The TIF32-CTD interacted in vitro with helices 16-18 of domain I in 18S rRNA, and the TIF32-NTD and NIP1 interacted with 40S protein RPS0A. These results suggest that eIF3 binds to the solvent side of the 40S subunit in a way that provides access to the interface side for the two eIF3 segments (NIP1-NTD and TIF32-CTD) that interact with eIF1, eIF5, and the eIF2/GTP/Met-tRNA(i)(Met) ternary complex.

/pdf/the-yeast-eif3-subunits-tif32-a-nip1-c-and-eif5-make-26xejxaq8m.pdf

The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5 make critical connections with the 40S ribosome in vivo

In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A ...

The hypusine-containing translation factor eIF5A.

Initiation factors IF2 in bacteria and eIF2 in eukaryotes are GTPases that bind Met-tRNA to the small ribosomal subunit. eIF5B, the eukaryotic ortholog of IF2, is a GTPase that promotes ribosomal subunit joining. Here we show that eIF5B GTPase activity is required for protein synthesis. Mutation of the conserved Asp-759 in human eIF5B GTP-binding domain to Asn converts eIF5B to an XTPase and introduces an XTP requirement for subunit joining and translation initiation. Thus, in contrast to bacteria where the single GTPase IF2 is sufficient to catalyze translation initiation, eukaryotic cells require hydrolysis of GTP by both eIF2 and eIF5B to complete translation initiation.

https://www.pnas.org/content/pnas/99/26/16689.full.pdf

Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation

The binding of eIF2–GTP–tRNAiMet ternary complex (TC) to 40S subunits is impaired in yeast prt1-1 (eIF3b) mutant extracts, but evidence is lacking that TC recruitment is a critical function of eIF3 in vivo If TC binding was rate-limiting in prt1-1 cells, overexpressing TC should suppress the temperature-sensitive phenotype and GCN4 translation should be strongly derepressed in this mutant, but neither was observed Rather, GCN4 translation is noninducible in prt1-1 cells, and genetic analysis indicates defective ribosomal scanning between the upstream open reading frames that mediate translational control prt1-1 cells also show reduced utilization of a near-cognate start codon, implicating eIF3 in AUG selection Using in vivo cross-linking, we observed accumulation of TC and mRNA/eIF4G on 40S subunits and a 48S ‘halfmer' in prt1-1 cells Genetic evidence suggests that 40S–60S subunit joining is not rate-limiting in the prt1-1 mutant Thus, eIF3b functions between 48S assembly and subunit joining to influence AUG recognition and reinitiation on GCN4 mRNA Other mutations that disrupt eIF2–eIF3 contacts in the multifactor complex (MFC) diminished 40S-bound TC, indicating that MFC formation enhances 43S assembly in vivo

/pdf/functions-of-eif3-downstream-of-48s-assembly-impact-aug-wlcwtj50hn.pdf

Byung-Sik Shin

Papers

eIF5A promotes translation of polyproline motifs.

The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5 make critical connections with the 40S ribosome in vivo

The hypusine-containing translation factor eIF5A.

Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation

Functions of eIF3 downstream of 48S assembly impact AUG recognition and GCN4 translational control.