Translation Initiation in Eukaryotes
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Translation Initiation in Eukaryotes
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Translation Initiation in Eukaryotes
- 1. Translation Initiation in Eukaryotes Zohaib Hussain Protein Structure and Function School of Materials and Science & Engineering (SMSE) BIo-Molecular & bio-Integrated Material (BIMIL) Laboratory
- 5. Translation Initiation in Eukaryotes • Highly regulated and rate-limiting process. • Assembly and disassembly of numerous transient and intermediate complexes • Over a dozen eukaryotic initiation factors (eIFs) • Process “culminates in the accommodation of a start codon marking the beginning of an open reading frame at the appropriate ribosomal site” • Contributions of structural biology
- 6. Translation Initiation in Eukaryotes Kummer, E., Leibundgut, M., Rackham, O., Lee, R. G., Boehringer, D., Filipovska, A., & Ban, N. (2018). Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM. Nature, 560(7717), 263.
- 7. Translation Initiation in Eukaryotes • Translation initiation is the target of regulation in a number of cellular processes including development, differentiation, stress response, and neuronal function. • Many diseases, including cancer and metabolic disorders, are connected with improper functioning or regulation of the initiation of protein synthesis. • Additionally, the cellular translation machinery can be hijacked by some viruses for their own reproduction
- 8. Aitken, C. E., & Lorsch, J. R. (2012). A mechanistic overview of translation initiation in eukaryotes. Nature structural & molecular biology, 19(6), 568- 576. https://doi.org/1 0.1038/nsmb.2303
- 9. Poulin F, Sonenberg N. Mechanism of Translation Initiation in Eukaryotes. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000- 2013. Available from: https://www.ncbi.nlm.nih .gov/books/NBK6597/
- 10. des Georges A, Dhote V, Kuhn L, Hellen CU, Pestova TV, et al. 2015. Structure of mammalian eIF3 in the context of the 43S preinitiation complex. Nature 525:491–95
- 11. Eukaryotic Initiation Factor 3 Complex • eIF3 is the initiation factor par excellence that can be found from the earliest steps all the way to the very latest steps of the initiation process. As the largest and most complex initiation factor, eIF3 is involved in virtually all steps of initiation
- 12. des Georges A, Dhote V, Kuhn L, Hellen CU, Pestova TV, et al. 2015. Structure of mammalian eIF3 in the context of the 43S preinitiation complex. Nature 525:491–95
- 13. The eIF2 Ternary Complex • The eIF2 TC is composed of eIF2 bound to a molecule of GTP, and it recruits the initiator methionylated Met-tRNA (tRNAi Met). eIF2 in turn is composed of three subunits: eIF2α, eIF2β, and eIF2γ.
- 15. The DHX29 Helicase • DHX29 is required for scanning on mRNAs with highly structured 5 UTSs that occur in higher eukaryotes. The silencing of DHX29 results in accumulation of free mRNA and the disassembly of polysome, symptomatic of initiation defects Hashem Y, des Georges A, Dhote V, Langlois R, Liao HY, et al. 2013a. Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29. Cell 153:1108–19
- 16. Hashem Y, des Georges A, Dhote V, Langlois R, Liao HY, et al. 2013a. Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29. Cell 153:1108–19
- 17. Hashem, Y., & Frank, J. (2018). The jigsaw puzzle of mRNA translation initiation in eukaryotes: a decade of structures unraveling the mechanics of the process. Annual review of biophysics, 47, 125-151.
- 18. Architecture of The Mammalian Translation Initiation Complex Kummer, E., Leibundgut, M., Rackham, O., Lee, R. G., Boehringer, D., Filipovska, A., & Ban, N. (2018). Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM. Nature, 560(7717), 263.
Editor's Notes
- Translation proceeds in three phases: Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon. Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the next codon. The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain. Termination: When a peptidyl tRNA encounters a stop codon, then the ribosome folds the polypeptide into its final structure.
- Animation of Translation: The elongation and membrane targeting stages of eukaryotic translation. The ribosome is green and yellow, the tRNAs are dark blue, and the other proteins involved are light blue. The produced peptide is released into the endoplasmic reticulum. Shape of the ribosom computed with something like Quantum Monte Carlo.
- In 2009, this pioneering work was awarded with the Nobel Prize in Chemistry The Nobel Prize in Chemistry 2009 was awarded jointly to Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath "for studies of the structure and function of the ribosome."
- Translation initiation in eukaryotes is a highly regulated and rate-limiting process. It results in the assembly and disassembly of numerous transient and intermediate complexes involving over a dozen eukaryotic initiation factors (eIFs). This process culminates in the accommodation of a start codon marking the beginning of an open reading frame at the appropriate ribosomal site. Although this process has been extensively studied by hundreds of groups for nearly half a century, it has been only recently, especially during the last decade, that we have gained deeper insight into the mechanics of the eukaryotic translation initiation process. This advance in knowledge is due in part to the contributions of structural biology, which have shed light on the molecular mechanics underlying the different functions of various eukaryotic initiation factors. In this review, we focus exclusively on the contribution of structural biology to the understanding of the eukaryotic initiation process, a long-standing jigsaw puzzle that is just starting to yield the bigger picture.
- The initiation of protein synthesis in eukaryotes involves at least twelve factors, compared to only three factors required for initiation in bacteria. This is mainly due to the more elaborate mRNA structure possessing a 5’-cap, a 3’-poly(A)-tail as well as long 5’-untranslated regions with secondary structure elements. Therefore, initiation factors are involved in binding and unwinding of the mRNA, in scanning of the 48S pre-initiation complex (PIC) for the start codon, and finally in determining the correct open reading frame.
- The initiation of protein synthesis in eukaryotes involves at least twelve factors, compared to only three factors required for initiation in bacteria. This is mainly due to the more elaborate mRNA structure possessing a 5’-cap, a 3’-poly(A)-tail as well as long 5’-untranslated regions with secondary structure elements. Therefore, initiation factors are involved in binding and unwinding of the mRNA, in scanning of the 48S pre-initiation complex (PIC) for the start codon, and finally in determining the correct open reading frame. Furthermore, translation initiation is the target of regulation in a number of cellular processes including development, differentiation, stress response, and neuronal function. Consequently, many diseases, including cancer and metabolic disorders, are connected with improper functioning or regulation of the initiation of protein synthesis. Additionally, the cellular translation machinery can be hijacked by some viruses for their own reproduction. In these cases, the ribosomes of the host organism directly bind to a secondary structure element in the viral mRNA termed internal ribosomal entry site (IRES), which allows initiation with a reduced set of initiation factors or entirely without the canonical initiation machinery.
- 1 Schematic of the translation initiation pathway in eukaryotes. Initiation begins with the formation of the ternary complex (TC) containing eIF2•GTP and the initiator tRNA (1). The ternary complex is recruited to the 40S subunit with the help of eIFs 1, 1A, 3 and 5 to form the PIC (2). Meanwhile, the mRNA is bound by the eIF4 factors and the PABP to form an activated mRNP (3a), which is then recruited to the PIC (3b). Once bound at the 5′ end of the mRNA, the PIC scans to locate the start (AUG) codon (4). Start codon recognition triggers eIF1 release and conversion of eIF2 to its GDP-bound state, arresting the scanning process (5). eIF2•GDP and eIF5 dissociate, clearing the way for eIF5B to mediate joining of the 60S subunit (6). Subunit joining is followed by GTP hydrolysis by eIF5B and factor dissociation to form the 80S initiation complex (IC) (7).
- formation of the 43S pre-initiation complex, when the Met-tRNAiMet is delivered by eIF2 to the P site of the 40S ribosomal subunit; recruitment of the 43S complex to the 5' end of the mRNA by eIF3 and the eIF4 factors; scanning of the 5' untranslated region (UTR) and recognition of the AUG codon, and assembly of the 80S ribosome Scanning model of eukaryotic translation initiation. The Met-tRNAiMet interacts with eIF2·GTP to form the ternary complex. The multifactor complex is an intermediate facilitating Met-tRNAiMet recruitment to the 40S ribosomal subunit, which generates the 43S complex. The eIF4 factors promote the recruitment of the 43S complex to the mRNA 5' end. The 43S complex then scans the mRNA 5' untranslated region to localize the initiator AUG (48S complex). It is not known whether the eIF4 factors participate in the scanning process. Base pairing between the Met-tRNAiMet anticodon and the AUG codon activates eIF2 GTPase, which causes the release of the bound factors. eIF1A and eIF5B interact to promote ribosomal subunits joining. 60S joining activates the GTPase activity of eIF5B, leading to its release from the 80S ribosome. The ribosome is then ready to accept the first elongating aminoacyl-tRNA in the A site. Initiation factors are labeled with their respective number, and ribosomal subunits are depicted as shaded ovals.
- Structure of the 80S eukaryotic ribosome and the rRNA expansion segments (ESs) of the small ribosomal subunit. (a) The eukaryote-specific ri bosomal features (in red ) compared to the bacterial ribosome. Small subunit (SSU) shown from (b) the solvent side and (c) the intersubunit face. Ribosomal proteins are shown as gray surface, and the 18S rRNA as yellow ribbons. (d,e) Involvement of ES6 and ES7 in the binding of eukaryotic initiation factor 3 (eIF3) as deduced by the cryo-electron microscopy structure of the 43S mammalian preinitiation complex (30). Additional abbreviation: LSU, large subunit.
- Structures of eIF3 fragments and subunits by X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy (cryo-EM). (a) Different fragments of eIF3 subunits are colored variably, and the accession codes are written in black below the name of each subunit. (b,c) Small subunit (SSU) shown in yellow surface, and eIF3 in colored ribbons. Different eIF3 subunits are labeled and colored variably. The displayed eIF3 structure is a model derived from an ∼6-A˚ cryo-EM map of the mammalian 43S preinitiation complex (30), which was in part interpreted thanks to the high-resolution structures of eIF3 fragments. Additional abbreviations: C-ter, C-terminus; eIF, eukaryotic initiation factor; h44, helix 44; RRM, RNA recognition motif.
- The eIF2 Ternary Complex The eIF2 TC is composed of eIF2 bound to a molecule of GTP, and it recruits the initiator methionylated Met-tRNA (tRNAi Met). eIF2 in turn is composed of three subunits: eIF2α, eIF2β, and eIF2γ. T Structures of the eIF2-TC fragments and subunits. (a) Different fragments of eIF2 subunits are colored variably, and the accession codes are written in black below the name of each subunit. (b) Structure of the TC seen from two different orientations. (c) The TC in the context of the SSU is shown in yellow surface, and eIF3 in colored ribbons. Different eIF3 subunits are labeled and colored variably. The conformation of the TC is a model derived from a ∼12-A˚ cryo-electron microscopy (cryo-EM) map, then confirmed by the ∼6-A˚ cryo-EM map of the mammalian 43S preinitiation complex (30, 49). (d,e) Blowup on the TC seen from two different orientations. Additional abbreviations: eIF, eukaryotic initiation factor; EM, electron microscopy; GDP, guanosine diphosphate; GTP, guanosine triphosphate; SSU, small subunit; TC, ternary complex; tRNA, transfer RNA.
- Figure 5 Model of the structure of the mammalian 43S preinitiation complex. (a) The 43S seen from the solvent side. Thick red dashed line reflects the continuity of eIF3a structure that remains unsolved at this region. (b) The 43S seen from the beak side. Transparent green surface reflects the shape of the N-terminal part of DHX29 that was not modeled. Black dashed circle indicates the binding site of eIF3j, and dashed red circle indicates the position of the dsRBD of DHX29. Notice that eIF3j and DHX29 dsRBD overlap on the same ribosomal position. Abbreviations: C-ter, C-terminus; dsRBD, DHX, DExH-box helicase; double-stranded RNA binding domain; eIF, eukaryotic initiation factor; h44, helix 44; RRM, RNA recognition motif; SSU, small subunit; tRNA, transfer RNA.
- Figure 6 Simplified model of eukaryotic translation initiation. The model is based on different structures of eukaryotic (pre)initiation complexes. Different stages of the process are indicated by the circled numbers. The thick, curved green arrow indicates the eIF3b-i-g module relocation from the solvent to the intersubunit side of the 40S subunit, and the red curved arrow indicates its relocation away from the intersubunit face, probably back to the solvent side, until the release of eIF3. Dark gray arrows indicate the conformational change of the eIF2 ternary complex upon eIF3b-i-g relocation and departure to and from the intersubunit face of the 40S, which may reflect its previously described PIN and POUT conformations. Abbreviations: ABCE1, ATP binding cassette E1; C-ter; C-terminus; eIF, eukaryotic initiation factor; GDP, guanosine diphosphate; mRNA, messenger RNA; Pi, inorganic phosphorous; RRM, RNA recognition motif; tRNA, transfer RNA.
- The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals3,4 . Furthermore, only one type of methionyl transfer RNA (tRNAMet) is used for both initiation and elongation4,5 , necessitating that the initiation factor specifically recognizes the formylated version of tRNAMet (fMet–tRNAMet). Lastly, most mitochondrial mRNAs do not possess 5′ leader sequences to promote mRNA binding to the ribosome2 . There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2 Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet–tRNAMet and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane.