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Translation Phases in Eukaryotes

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Part of the Methods in Molecular Biology book series (MIMB,volume 2533)

Abstract

Protein synthesis in eukaryotes is carried out by 80S ribosomes with the help of many specific translation factors. Translation comprises four major steps: initiation, elongation, termination, and ribosome recycling. In this review, we provide a comprehensive list of translation factors required for protein synthesis in yeast and higher eukaryotes and summarize the mechanisms of each individual phase of eukaryotic translation.

Key words

  • Translation
  • Ribosome
  • mRNA
  • tRNA
  • Yeast

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1 Introduction

In all domains of life, information encoded in mRNA is translated to protein by a supramolecular machine called ribosome. Eukaryotic ribosome consists of a small subunit (40S ) and a large subunit (60S ) that together form the 80S ribosome. The ribosome reads the information one codon (three nucleotides) at a time using aminoacyl-tRNAs as adaptor molecules that recognize each codon to insert the appropriate amino acid. Ribosomes from bacteria , archaea , and eukarya share a high degree of sequence and structure conservation, indicating a common evolutionary origin. Furthermore, they share a similar central core where mRNA decoding, peptidyl transfer, and translocation of tRNA and mRNA by one codon take place. The process of translation can be divided into four main phases: initiation , elongation , termination , and ribosome recycling . During the initiation phase, eukaryotic translation initiation factors (eIFs) promote the assembly of 80S ribosomes at the AUG start codon with an initiator methionyl-tRNA bound to the P site (Table 1). During elongation , 80S ribosomes move processively along the mRNA , synthesizing the encoded protein through the coordinated actions of aminoacyl-tRNAs and the eukaryotic translation elongation factors (eEFs) (Table 2). At the end of the open reading frame, the ribosome encounters a termination codon recognized by eukaryotic release factors (eRFs) (Table 2), which promote the release of the nascent protein from the ribosome. Finally, during the recycling phase, the ribosome complex is recycled to the 40S and 60S subunits to begin a new round of translation . In this review, we describe the four phases of eukaryotic translation and function of translation factors (Tables 1 and 2). In the accompanying methods chapter, we focus on in vitro reconstitution of initiation and elongation phases of translation . Detailed aspects of translation termination and recycling are discussed in recent reviews [1, 2].

Table 1 Eukaryotic translation initiation factors
Table 2 Eukaryotic translation elongation and termination factors

2 Initiation

The initiation phase leads to the formation of 80S ribosomes where the initiator tRNA (Met-tRNAi Met) and the start codon are positioned in the P site. This process requires at least ten eukaryotic initiation factors, eIFs (Table 1), and comprises two major steps. In the first step, Met-tRNAi Met binds to the start codon in the P site of the 40S subunit to form the 48S initiation complex. In the second step, the 60S subunit joins the 48S complex to form the 80S initiation complex that is ready for elongation (Fig. 1) [3, 4].

Fig. 1
figure 1

Translation initiation . eIF2–GTP–Met-tRNAi Met binds to the 40S ribosomes in presence of eIF1, eIF1A, eIF3, and eIF5 to form 43S PIC . Then mRNA is recruited with the help of eIF4F complex to form the 48S PIC . After mRNA scanning and recognition of the start codon, the 60S subunit joins with the help of eIF5B to form the 80S IC [4, 59, 60]

Formation of 48S preinitiation complex . During this step, eIF3, eIF1, and eIF1A are recruited to 40S subunits, followed by subsequent attachment of eIF5 and eIF2–GTP–Met-tRNAi Met to form 43S complexes. eIF3 binds to the 40S subunit side that faces the solvent in the 80S ribosome [5], whereas eIF1 binds between the platform of the 40S subunit and Met-tRNAi Met to the 40S side that will form the interface to the 60S subunit [6]. The structured domain of eIF1A resides in the A site, forming a bridge over the mRNA channel, and its N- and C-terminal tails extend into the P site. Binding of eIF1 and eIF1A to the 40S subunit induces conformational changes [7, 8], which result in the opening of the mRNA entry channel and the establishment of a new connection between the head and body domains of the 40S subunit on the solvent side between helix 16 of 18S rRNA and the ribosomal protein uS3 . Although 43S complexes can bind model mRNAs with completely unstructured 5′ UTRs in the absence of eIFs [9], attachment of natural mRNAs requires a coordinated activity of eIF4F protein complex, which unwinds the 5′ cap-proximal region of mRNA . Following mRNA attachment, the 43S complex scans the mRNA downstream of the cap in search for the initiation codon. During the scanning process, the secondary structures in the 5′ UTR unwind and the 43S complex moves until the initiation codon is recognized. eIF1 and eIF1A play key roles in the 43S complex formation and scanning; omission of either or both reduces or suppresses the scanning ability, indicating that the movement of 43S complexes requires the scanning-competent conformation induced by eIF1 and eIF1A [7]. eIF3, which is indispensable for 48S complex formation, forms an extension of the mRNA-binding channel that might contribute to scanning [10]. The fidelity of start codon recognition is ensured by the discriminatory mechanism that promotes recognition of the correct initiation codon and prevents premature Met-tRNAi Met landing at near-cognate triplets in the 5′ UTR. The bona fide start site is usually the first AUG triplet in an optimum nucleotide context GCC(A/G)CCAUGG, with a purine at the −3 and a G at the +4 position relative to A of the AUG codon [11]. eIF1 has an important role in initiation codon recognition: it enables 43S complexes to select the correct AUG in a poor initiation context or an AUG located within 8 nucleotides of the mRNA 5′ end, and promotes dissociation of the ribosomal complexes that aberrantly assemble at such triplets in the absence of eIF1 [9, 12, 13]. Following start codon recognition and GTP hydrolysis, Pi is released from eIF2–GTP + Pi and eIF1 dissociates from the ribosome [14,15,16].

Ribosomal subunit joining . eIF5B, a ribosome-associated GTPase , facilitates the joining of the 60S subunit and the dissociation of eIF1A, eIF5, and eIF2–GDP [17]. GTP hydrolysis by eIF5B is essential for its own release from assembled 80S ribosomes . eIF5B occupies the region in the intersubunit cleft [18] and promotes subunit joining by burying large solvent-accessible surfaces on both subunits [19]. After the 60S subunit joining and dissociation of initiation factors, the 80S initiation complex with the anticodon of Met-tRNAi Met in the P site base-paired with the start codon becomes elongation competent.

3 Elongation

The elongation phase comprises three steps: decoding of mRNA codons by the cognate aminoacyl-tRNAs , peptide bond formation, and translocation of the tRNA–mRNA complex, resulting in movement of peptidyl-tRNA from the A site to the P site and presenting the next codon in the A site [1] (Fig. 2). Four elongation factors are required the three steps (Table 2).

Fig. 2
figure 2

Translation elongation . During decoding, eEF1A delivers aa-tRNA to the A site. After peptide bond formation, the A-site peptidyl-tRNA and P-site tRNA are translocated to the P and E sites, respectively, with the help of eEF2. Deacylated tRNA is released from the E site and the elongation cycle repeats until a stop codon is reached. In yeast , an additional factor eEF3 is required during elongation [1]

The eukaryotic elongation factor eEF1A binds aminoacyl-tRNA in a GTP-dependent manner and delivers the aa-tRNA to the A site of the ribosome (Fig. 2). Codon recognition by the aa-tRNA triggers GTP hydrolysis by eEF1A, releasing the factor and enabling the aa-tRNA to accommodate in the A site, presumably following the same pathway as described for bacterial ribosomes [20,21,22,23,24]. However, decoding appears to take place more slowly and accurately than in bacteria , particularly in higher eukaryotes [25]. eEF1A is a member of the GTPase superfamily that binds and hydrolyzes GTP. The dissociation of GDP from eEF1A is accelerated by a guanine nucleotide exchange factor (GEF), eEF1B, which is composed of two subunits, eEF1Bα and eEF1Bγ in yeast , the first one containing the catalytic domain necessary for nucleotide exchange. eIF5A, which was originally identified as initiation factor, functions globally in translation elongation , especially when ribosome encounter polyproline sequences [2].

Following the accommodation of the aminoacyl-tRNA into the A site, the peptide bond formation with the P-site peptidyl-tRNA occurs rapidly in the peptidyl transferase center (PTC) . The PTC consists mainly of conserved rRNA elements of the 60S subunit that position the substrates for catalysis. Structural studies have revealed that the rRNA structure of the PTC is nearly superimposable between the eukaryotic and bacterial ribosomes [26,27,28], supporting the notion that the mechanism of peptide bond formation is universally conserved.

The pretranslocation complex formed as a result of peptide bond formation is dynamic and can spontaneously fluctuate between several conformations, in prokaryotes and eukaryotes alike [29, 30]. The ribosomal subunits undergo a ratchet-like motion from the so-called classical to rotated state triggering movement of the tRNAs to adopt hybrid states. In this state, the anticodon ends of the tRNAs remain positioned in the P and A sites of the 40S subunit, while the tRNA acceptor ends are positioned in the E site and P site of the 60S subunit (P/E and E/P states, respectively) [31,32,33]. Translocation of the mRNA –tRNA in the ribosome requires elongation factor eEF2, which facilitates the return of hybrid tRNAs to the classical state, E/E and P/P (canonical E and P sites). eEF2 in its GTP-bound state facilitates and stabilizes the hybrid rotated state. Conformational changes in eEF2 upon GTP hydrolysis and Pi release unlock the ribosome allowing tRNA and mRNA movement and then lock the subunits in the posttranslocation state. In that state of the ribosome, a deacylated tRNA occupies the E site and the peptidyl-tRNA is in the P site, leaving the A site vacant and available for binding of the next aminoacyl-tRNA in complex with eEF1A [34, 35]. In addition to eEF1A and eEF2, a third factor, eEF3, is essential for elongation in fungi [36]. The elongation cycle is repeated until a complete protein has been synthesized and a stop codon is encountered by the ribosome.

4 Termination

Termination occurs when the ribosome reaches a stop codon (UAA , UGA , or UAG ) [1, 37,38,39] (Fig. 3). Termination is catalyzed by two protein factors, eRF1, which recognizes all three stop codons , and the GTPase eRF3, which facilitates termination at a cost of GTP hydrolysis [39,40,41]. eRF1 contains a structural Asn-Ile-Lys-Ser (NIKS) motif and several other conserved elements, including the Gly-Thr-Ser (GTS) and YxCxxxF motifs, which are involved in the recognition of the termination codons [42,43,44,45,46]. In addition, the central domain of eRF1 contains a Gly-Gly-Gln (GGQ) motif that extends into the peptidyl-transferase center to promote peptidyl-tRNA hydrolysis and the release of nascent peptide [47,48,49,50]. eRF1 requires eRF3 (a specialized GTPase ) for proper function (Table 2). eRF1 and eRF3 bind to one another with very high affinity and probably enter the ribosome as a complex [51]. GTP hydrolysis positions the GGQ region of eRF1 in the peptidyl transferase center and triggers peptidyl-tRNA hydrolysis. eRF3 strongly enhances peptide release by eRF1 in the presence of GTP, but not GDP or nonhydrolyzable GTP analogs [39]. Moreover, other trans-acting factors are known to affect translation termination . ABCE1 (Rli1 in yeast ) interacts with eRF1 to stimulate its catalytic activity by stabilizing the active conformation of eRF1 [52,53,54,55].

Fig. 3
figure 3

Translation termination and recycling. Termination occurs when a stop codon enters the A site of the ribosome and is catalyzed by eRF1 and eRF3. Peptide release is promoted by ABCE1 which also induces subunits dissociation [1]

5 Ribosome Recycling

During recycling the ribosomal subunits dissociate and the mRNA together with the deacylated tRNA are released to regenerate the necessary components for subsequent rounds of translation . Recycling of ribosomal subunits is achieved by the ATPase ABCE1 (Rli1 in yeast ), an essential protein which contains two nucleotide-binding domains and an amino-terminal iron–sulfur cluster (Fe-S) and induces subunits dissociation at the cost of ATP hydrolysis [53, 54, 56]. Upon binding and hydrolysis of ATP , the Fe-S cluster undergoes a conformational change that drives eRF1 into the ribosomal intersubunit space, leading to dissociation of posttermination ribosomes into 40S and 60S subunits. Deacylated tRNA and mRNA are then released from the 40S subunits, which may be additionally promoted by Ligatin (eIF2D) [57] (Table 2). Initiation factors eIF1, eIF1A, and eIF3j can also promote tRNA and mRNA dissociation from the 40S subunit in vitro [54]. Following termination , in some cases full dissociation of the ribosomal complex will occur, whereas in other cases partial dissolution of the complex will allow for reinitiation, a term used to describe a process wherein ribosome translates two or more ORFs in a transcript without undergoing complete recycling between these events [58]. However, the mechanism of reinitiation is not fully understood.

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Acknowledgments

We thank Prof. Marina Rodnina for critical reading of the manuscript. This work is supported by the Deutsche Forschungsgemeinschaft (DFG) in the framework of the Schwerpunktprogram (SPP1784), and by the Max Planck Society.

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Blanchet, S., Ranjan, N. (2022). Translation Phases in Eukaryotes. In: Entian, KD. (eds) Ribosome Biogenesis. Methods in Molecular Biology, vol 2533. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2501-9_13

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  • DOI: https://doi.org/10.1007/978-1-0716-2501-9_13

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