Translation is a cyclical process in which ribosomal subunits that participate in initiation are derived by recycling of post-termination ribosomal complexes (post-TCs) that have completed the previous round of translation. Post-TCs first dissociate into free 60S subunits and 40S subunits bound to mRNA and with a P-site deacylated tRNA. Release of tRNA and mRNA from recycled 40S subunits requires eukaryotic initiation factor 3 (eIF3), eIF1 and eIF1A. Deacylated tRNA, which is removed from the P-site of the ribosomal 40S subunit, is replaced by Met-tRNAMeti, which binds as a ternary complex with eIF2-GTP.
Initiation on most eukaryotic mRNAs involves scanning by ribosomal 43S preinitiation complexes on the 5′ untranslated region (UTR) from the 5′ cap-proximal point of initial attachment to the initiation codon. The mechanism of scanning is incompletely understood, but requires an 'open' conformation of the 40S subunit, which is induced by eIF1 and eIF1A, is coupled to the activities of the DEAD-box RNA helicase eIF4A, its cofactor eIF4B and eIF4G, and may involve additional DEAD box- or DExH box-containing proteins such as DExH box protein 29 (DHX29; higher eukaryotes) and DEAD box helicase 1 (Ded1; yeast).
In a current model for initiation codon recognition, establishment of codon–anticodon base pairing is accompanied by displacement of eIF1 from the P-site, which switches the 40S subunit to a closed conformation that is locked onto mRNA and relieves repression of eIF5-induced hydrolysis of eIF2-bound GTP and Pi release. eIF5B then mediates the dissociation of eIF2–GDP, eIF1 and eIF1A from the interface surface of the 40S subunit and the joining to it of a 60S ribosomal subunit, to form an 80S ribosome with initiator tRNA in the P-site.
Regulation of eIF activity by reversible phosphorylation affects most mRNAs that are translated by the canonical scanning-dependent mode of initiation. The best-characterized examples are phosphorylation of mammalian eIF2 by any of four stress-activated kinases (which leads to a reduction in the level of eIF2–GTP–Met-tRNAMeti ternary complexes) and of mammalian 4E-binding proteins, mainly by mTOR (in response to mitogens and growth factors), which releases eIF4E for assimilation into eIF4F and promotes translation.
Sequence-specific RNA-binding proteins have the potential to selectively regulate specific mRNAs or classes of mRNA. 3′ UTR-binding proteins commonly repress translation by forming an inhibitory closed loop with a 5′ cap-binding protein (which may be eIF4E) and an intermediate bridging protein, whereas poly(A)-binding protein (PABP) bound to the 3′ poly(A) tail enhances initiation, possibly by tethering eIF4F so that it is available, despite dissociation from the 5′ cap, to promote further rounds of initiation on the same mRNA without having to be recruited de novo from solution.
MicroRNAs have recently emerged as important regulators of translation that also act by binding to the 3′ UTR. The mechanism of repression has not yet been fully elucidated, but appears to have two components, both mediated by the carboxy-terminal domain of the protein GW182: true repression of translation and an accelerated rate of deadenylation-dependent mRNA degradation.
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).
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We thank J. Doudna, A. Marintchev and L. Passmore for figures. Research in the authors' laboratories is supported by grants from the Biotechnology and Biological Sciences Research Council and The Wellcome Trust (RJJ), the National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (CUTH) and the NIH National Institute of General Medical Sciences (TVP).
Tatyana V. Pestova and Christopher U. T. Hellen have filed a patent on DHX29 and its use to identify therapeutic regulators of gene expression.
Supplementary information S2 (box) | Reinitiation after translation of a short upstream ORF (uORF); instructive differences between yeast and vertebrates. (PDF 263 kb)
Supplementary information S4 (box) | Similarities and differences between the mechanisms of regulation of yeast GCN4 and mammalian ATF4/5 mRNAs (PDF 253 kb)
Supplementary information S5 (box) | Does the "closed loop" configuration resulting from PABP-eIF4G interaction promote ribosome recycling? (PDF 241 kb)
The unique initiator tRNA, aminoacylated with methionine, that is used to initiate protein synthesis. Its anticodon is complementary to the AUG initiation codon; it forms a specific ternary complex with eIF2 and GTP and it binds to the ribosomal P-site.
The site on a ribosome that holds the tRNA that is linked to the growing peptide chain (peptidyl-tRNA).
- Internal ribosome entry site
A structure that is located in the 5′ UTR or ORF of some mRNAs of cellular or viral origin. It mediates translation initiation independently of the 5′ end of mRNA by recruiting the ribosome directly to an internal position on the mRNA.
- ATP-binding cassette (ABC) family
A family of proteins that typically contain two nucleotide-binding domains (NBDs), which form two composite nucleotide-binding sites. The transition between the closed ATP-bound and open ADP-bound states induces a tweezer-like powerstroke between NBDs, which causes conformational changes in associated domains and/or macromolecules.
- 18S rRNA
Ribosomal RNA of the 40S ribosomal subunit. It determines the overall shape of the 40S subunit and is the main component of its decoding centre. It is also involved in formation of the main contacts between 40S and 60S ribosomal subunits.
The site on a ribosome that holds the new, incoming aminoacyl-tRNA.
The site on a ribosome that accommodates deacylated tRNA before it is released from the ribosome.
- DEAD-box RNA helicase
An RNA helicase that contains the DEAD (Asp-Glu-Ala-Asp) or DExD/H (Asp-Glu-X-Asp,His; where X represents any amino acid) motifs. These proteins use the energy of ATP hydrolysis to unwind RNA.
- RRM domain
(RNA-recognition motif). A protein domain that contains two short consensus sequences embedded in a structurally conserved region of ∼80 amino acids.
- HEAT domain
A protein domain of 37–47 amino acids that consists of tandemly repeated pairs of antiparallel α-helices. HEAT domains have a superhelical structure and often function as protein–protein interaction surfaces.
- GTPase-activating protein
A protein that stimulates the intrinsic ability of a GTPase to hydrolyse GTP to GDP.
- Arginine finger
A catalytic residue that was first defined for RasGAPs, and that supplies a catalytic arginine residue into the active site of Ras to increase the reaction rate.
A small RNA of ∼ 21 nucleotides that regulates the expression of mRNAs with which it is partially complementary in sequence.
- KH domain
(K-homology domain). A protein domain, originally identified in the human hnRNP-K protein, that is important for RNA binding and probably binds RNA directly.
A family of proteins that are characterized by the presence of two homology domains: PAZ and PIWI. These proteins are essential for diverse RNA silencing pathways.
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Jackson, R., Hellen, C. & Pestova, T. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11, 113–127 (2010). https://doi.org/10.1038/nrm2838
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