The mechanism of eukaryotic translation initiation and principles of its regulation

Abstract

Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control of gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions in ribosomal initiation complexes, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity (which affects almost all scanning-dependent initiation) and through sequence-specific RNA-binding proteins and microRNAs (which affect individual mRNAs).

Figure 1. Model of the canonical pathway of eukaryotic translation initiation

This pathway is divided into eight stages (2–9), which follow (1) recycling of post-termination complexes to yield separated 40S and 60S ribosomal subunits, and result in formation of an 80S ribosomal initiation complex in which Met-tRNA^Met_i is base-paired with the initiation codon in the ribosomal P-site and which is competent to start the elongation stage of translation. These stages are: (2) formation of the eIF2•GTP/Met-tRNA^Met_i ternary complex; (3) formation of a 43S preinitiation complex comprising a 40S subunit, eIF1, eIF1A, eIF3, eIF2•GTP/Met-tRNA^Met_i and probably eIF5; (4) mRNA activation, during which the mRNA cap-proximal region is unwound in an ATP-dependent manner by eIF4F with eIF4B; (5) attachment of the 43S complex to this mRNA region; (6) scanning of the 5’UTR in a 5’→3’direction by 43S complexes; (7) recognition of the initiation codon and 48S initiation complex formation, which switches the scanning complex to a ‘closed’ conformation and leads to displacement of eIF1, permitting eIF5-mediated hydrolysis of eIF2-bound GTP and Pi release; (8) joining of 60S subunits to 48S complexes and concomitant displacement of eIF2•GDP and other factors (eIF1, eIF3, eIF4B, eIF4F and eIF5) mediated by eIF5B; and (9) GTP hydrolysis by eIF5B and release of eIF1A and eIF5B•GDP from assembled elongation-competent 80S ribosomes. Translation is a cyclical process in which termination follows elongation, and leads to recycling (1) which generates separated ribosomal subunits. The model omits potential ‘closed-loop’ interactions involving poly(A) binding protein (PABP), eukaryotic release facto 3 (eRF3) and eIF4F during recycling (Supplementary information S5), and recycling of eIF2•GDP by eIF2B. Whether eRF3 is still present on ribosomes at the recycling stage is unknown.

Figure 2. Architecture of ribosomal initiation complexes

(A) Model of a 40S subunit with eIF3 on its exterior (solvent) surface and eIF4G bound to eIF3 near the E-site, based on cryoelectron microscopy analysis, and showing positions of mRNA (red line) and eIF1 on the subunit interface. Binding of eIF3 to the solvent surface is compatible with its potential partial retention on ribosomes during translation of short upstream open reading frames (uORFs). Adapted with permission from Ref. . (B) Positions of eIF1 (magenta) and eIF1A (with its structured domain in light blue , its carboxy-terminal tail in dark blue and its amino-terminal tail in green) on the 40S subunit relative to mRNA (red) and P-site tRNA (yellow), based on directed hydroxyl radical probing data^, and modeled using T. thermophilus 30S subunit crystal structures (PDB codes 1JGO and 1JGP). (C) Cryoelectron microscopy reconstructions of yeast apo 40S subunits (left panel) and 40S–eIF1–eIF1A complexes (right panel), labelled to indicate the A-, P- and E-sites in the mRNA-binding channel, and the positions of rRNA helices h16, h18 and h34, which are involved in forming the mRNA entry-channel (h18-h34) and the eIF1- and eIF1A–induced head–shoulder connection (h16-rpS3) (indicated by an asterisk). Adapted from Ref. with permission.

Figure 3. eIF4G domain structure, interactions, and its position in a scanning 43S complex

(A) Schematic representation of the longest isoform of eIF4GI (Genbank Acc. NP_937884), of its p100 (C-terminal two-thirds) and p50 (central one-third) fragments, and of p97, showing binding sites for SLBP-interacting protein 1 (SLIP1), PABP, eIF4E, eIF4A, eIF3, and MAP kinase interacting Ser/Thr kinase 1 (Mnk1) or Mnk2 and for RNA (dotted lines below eIF4G1). The interactions of eIF4G with eIF4E and Mnk1 are required for phosphorylation of eIF4E by Mnk1; interactions of eIF4G with PABP and SLIP1 tether eIF4F to the 3’-end of mRNA (see text). The amino acid residues at the N-termini of the PABP-binding domain (PAM-1), 4E–BR (eIF4E–binding domain) and HEAT-1 (also known as MIF4G), HEAT-2 (also known as MA3) and HEAT-3 (also known as W2) domains are indicated, as is the cleavage site in eIF4GI for the picornavirus proteinase 2A^pro (Supplementary information S3), which divides eIF4G into an N-terminal domain that binds eIF4E and PABP, and a C-terminal domain that provides all functions of eIF4G required for initiation on Type 1 and Type 2 IRESs (see Box 1). This cleavage event contributes to the switch from host to viral translation during many picornavirus infections (see Supplementary Information S3). (B) Hypothetical model of the scanning 43S preinitiation complex, viewed from the solvent face, showing associated factors and domains of factors, including eIF4E, eIF4G’s 4E–BR, HEAT-1, HEAT-2 and HEAT-3 domains of eIF4G, the C-terminal and RRM domains of eIF4H and the N-terminal and C-terminal domains of eIF4A. The direction of scanning (5’→3’) is shown by an arrow, and in this model, eIF4A is on the leading (3’) side of the scanning complex. Adapted from Ref. with permission.

Figure 4. The mechanism of regulation of ATF4 and ATF5 mRNA translation

(a) Diagram shows the sizes, spacing and disposition of the two upstream open reading frames (uORFs) in human, mouse, rat, cow and chicken activating transciption factor 4 (ATF4) mRNAs and the four mammalian ATF5 mRNAs^, . (b) The pattern of translation in control (unstressed) conditions when eIF2-GTP-Met-tRNA_i ternary complexes (eIF2-TCs) are abundant. Small (40S) ribosomal subunits with associated eIF2-TCs (blue) scan the mRNA in the direction shown by the short horizontal arrows, and nascent protein chains are shown by the black zig-zag line associated with the large (60S) ribosomal subunit. If eIF2-TCs are abundant, most of the 40S subunits that resume scanning after uORF1 translation will acquire a new eIF2-TC in time to initiate translation of uORF2, and ribosomes that translate this second uORF will be unable to initiate at the ATF4/5 AUG because uORF2 is rather too long to allow rescanning, and also because it would require backwards scanning which doesn’t appear to occur over significant distances. (c) Pattern of translation in stressed conditions (for example, thapsigargin treatment), when eIF2-TC availability is low due to eIF2 phosphorylation by activated PERK. Consequently, most of the 40S subunits that resume scanning after translating uORF1 acquire a new eIF2-TC only after they have passed the uORF2 initiation codon, but in time to initiate at the next AUG which is at the start of the ATF ORF in both cases.

Figure 5. Models of miRNA-mediated repression of translation of target mRNAs

(a) Examples of the imperfect complementarity between miRNAs (boxed) and their mRNA target sites (upper line) for two validated C. elegans miRNA/mRNA interactions. The interaction typically involves perfect contiguous base-pairing of miRNA residues 2–8 (the seed match), in some cases extending to residues 1–9, followed by mismatch bulges in either the miRNA or mRNA (or both), and then irregular base-pairing of the miRNA 3’-end to the mRNA. (b) Schematic depiction of the different mechanisms by which miRNAs might regulate their target mRNAs. For clarity only a single miRNA target site is shown, and the other proteins in the complex with Ago and GW182 (the most downstream effector of repression identified so far) have been omitted. (c) Tethering experiments showing repression by tethered Ago or GW182. The 3’-UTR of the reporter mRNA has multiple bacteriophage lambda Box B motifs, or bacteriophage MS2 high affinity sites for coat protein; the test protein (Ago or GW182) is expressed as a fusion with an epitope tag (blue), to allow monitoring of expression levels, and either lambda N-peptide (red) or MS2 coat protein (green). Controls have the epitope tag but lack N-peptide or MS2 coat protein sequences. Tethering a translational activator to the 3’-UTR by the same method results in stimulation of translation, e.g. tethering PABP to a poly(A)- mRNA.

Box 1

IRES-mediated translation initiation

Box 2

Generic model for the regulation of initiation by 3’ UTR-protein interactions