What is translation? 2018-01-25T11:00:58+00:00

What is translation?

Translation is a process that involves the synthesis of an amino acid chain from an mRNA blueprint. These polypeptide chains fold into functional proteins. Translation occurs outside the nucleus once nuclear processing of the pre-mRNA is complete and the mRNA molecules have been transported to the cytoplasm via nuclear pores. Translation is primarily facilitated by ribosomes located on the rough endoplasmic reticulum, on the outer surface of the nuclear envelope, or in the cytoplasm.

The four steps of translation are:
  1. Initiation
  2. Elongation
  3. Termination
  4. Recycling
The general principles of translation are similar between prokaryotes and eukaryotes, however specific details may vary significantly. Here, we focus on translation mechanisms in eukaryotes.

A number of molecular components play a role in translation, the most prominent being the ribosome. This macromolecular complex is made up of multiple proteins and rRNA molecules. All ribosomes feature a small and a large subunit, however the makeup of these subunits differs substantially between species. In humans, for example, the small 40S subunit is composed of 33 proteins and a single 18S rRNA molecule while the large 60S subunit is composed of 47 proteins and three rRNAs (5S, 5.8S and 28S) [1].

Despite the identification of 80 proteins associated with the human ribosome, only 34 are found in other eukaryotes or prokaryotes [2]. While general functions have been proposed to ribosome associated proteins, such as stabilization of the complex and regulation of translation, some have also been attributed to the co-translational modification of newly synthesized proteins (reviewed in [3]).

Elongation

In contrast to the initiation, termination and ribosome recycling stages of translation, the mechanisms that drive elongation are highly conserved between eukaryotes and bacteria (reviewed in [4]).

Codon recognition

Elongation occurs over several well-defined steps, beginning with the recognition of the mRNA codons by their corresponding aminoacyl-tRNA. Association with the mRNA occurs via the ribosomal A site and is influenced by various elongation factors. For example, the GTPase eEF1A delivers aminoacyl-tRNAs to the A site after being activated by eEF1B, a guanine nucleotide exchange factor (GEF) that accelerates the dissociation of GDP from eEF1A [5].

Peptide bond formation

Following mRNA codon recognition, a peptide bond is created between the aminoacyl-tRNA and the peptidyl-tRNA (which is located in the ribosomal P site). This reaction is facilitated by peptidyl transferase, which itself is not a protein, but a highly conserved ribosomal RNA [5]. The mechanism of peptide bond formation involves conformational changes at the active site rather than chemical catalysis by ribosomal groups [6][7] and is driven by favorable entropy change [8].

Translocation of the mRNA and tRNAs through the ribosome

Once the peptide bond has formed, the A-site is made vacant when the peptidyl-tRNA occupying it moves into the P-site of the large ribosomal subunit, concomitantly replacing an existing deacylated tRNA which moves to the E-site before exiting the ribosome [9]. As the amino acid chain grows, and the A and P sites are transiently occupied by new aminoacyl- and peptidyl-tRNAs respectively, a translocation of the mRNA through the ribosome occurs.

Two mechanisms, which are characterized by conformational changes in the ribosomal subunits, facilitate mRNA and tRNA translocation. These are known as ‘ratcheting’ and ‘swiveling’.

Ratcheting is observed at all stages of translation, and sees the small ribosomal subunit undergoing a slight rotation, of approximately ~8° relative to the large subunit (reviewed in [10][9]). This is distinct from swiveling which involves a movement of the head domain (30S) of the small subunit. Importantly, swiveling plays a role in the intrinsic helicase activity of the ribosome, which is important for unwinding of secondary mRNA structures [11][12].

These mechanisms ultimately ensure the tRNA moves in a sequential (A-site to P-site to E-site) manner, and enable the formation of the intermediate states that are known to exist during mRNA-tRNA translocation. These states, which are also known as hybrid states [13], can be described using the eukaryotic model as an example. Here, the 3’ ends of the tRNA occupying the A and P sites move to occupy the P and E sites in the 60S subunit, while the 5’ ends, which are associated with the mRNA, remain anchored to the A and P sites of the 40S subunit respectively [13][9].

These hybrid states are momentarily stabilized by the binding of eEF2-GTP (EF-G – GTP in prokaryotes) to the ribosomal A-site. Hydrolysis of the GTP however, which is mediated through the GTPase activity of the EF-G or eukaryotic homolog eEF2, allows the ratcheting mechanism to continue, and causes the mRNA and the 5’ ends of the tRNA to move from the A and P sites into the P and E sites respectively. Once the canonical A/A, P/P, E/E (60S/40S) conformation is restored, the EF-G – GDP dissociates from the ribosome, leaving the A site open to receive a new aminoacyl-tRNA molecule [14][13].

GTP hydrolysis by EF-G / eEF2, and the subsequent swivelling of the head domain further assists in tRNA translocation by preventing any spontaneous backward movement of tRNA [15].

Initiation of Translation

The first step in translation is known as initiation. Here, the large (60S) and small (40S) ribosomal units are assembled into a fully functional 80S ribosome. This is positioned at the start codon (AUG) of the mRNA strand to be translated (reviewed in [16]).

Initiation is considered to be the rate limiting step in the overall process and is primarily regulated, and coordinated, by a group of proteins known as eukaryotic initiation factors (eIFs) [17]. These factors range in size and complexity; from the single 113kDa eIF1 subunit to the 700kDa eIF3 complex. In humans, at least 12 eIFs function in concert to regulate initiation, each playing a distinct role, which have been reviewed extensively in [18].

Initiation starts with the formation of a ternary complex that comprises eIF2, GTP and the initiator tRNA (Met-tRNAi). The primary role of the ternary complex is to deliver the initiator to the 40S subunit which subsequently establishes a 43S complex, also called the PIC (pre-initiation complex). With the assistance of eIF4G and eIF3, the PIC binds at or close to the 5’-terminus of mRNA. This is marked by a 7-methylguanosine cap (m7-G-cap). Once bound, the PIC scans the 5’ untranslated region to locate the initiation codon [16].

The leader sequence of the 5’-terminus is maintained in an unwound state by the helicase activity of several eIFs (including eIF4F, eIF4G, eIF4A, eIF4B, eIF3). Once the 40S subunit is positioned at the initiation codon, the 60S subunit is recruited to form an elongation-competent 80S ribosome. At this point the mRNA start codon is localized in the ribosomal P site, and the whole initiation complex is ready to enter the elongation phase [16].

It is important to note that the 40S subunit may bind to mRNA independently of the m7-G-cap. The most prominent example of this, which is believed to occur in 5-10% of cellular mRNAs, involves the 40S subunit binding to an internal ribosome entry site (IRES) [19]. Other m7-G-cap independent initiation pathways include shunting [20], tethering [21], translation enhancers [22], a TISU element [23], and a poly-adenylate leader in the 5’-terminus [24].

Ribosome Recycling

The final step in translation is ribosome recycling, which sees the ribosome split into its smaller subunit parts and prepare for another round of translation. In eukaryotes this means the 80S ribosome splits into its 40S and 60S subunits. Although this step marks the completion of the translation process, it may also occur for a variety of other reasons, including when synthesis of the polypeptide chain fails, when damaged mRNA is encountered, or following the assembly of empty ribosomes. Furthermore, this step is often described as the beginning of initiation, with the key protein that facilitates ribosome splitting, also associating with several initiation factors (ABCE1 has been shown to associate with eIF2, eIF3 and eIF5 in yeast models [25]).

In eukaryotes, ribosome recycling is primarily facilitated by ABCE1 (Rli1 in yeast), which is a member of the ABC superfamily of ATPases. This protein, which possesses two nucleotide binding domains and a unique FeS1 cluster domain, binds to the post-termination complex once the RF3-GDP has dissociated from the ribosome. This association forms through the interaction between the FeS cluster and eRF1. Importantly, ACBE1 also contains numerous binding sites that permit interactions between ribosomal subunits, and various ribosomal proteins. For example a HLH motif in the first nucleotide binding domain has been shown to bind to the 18S rRNA as well as rpS24-A [26]. Although the exact mechanism that drives ribosomal splitting remains unclear, it is proposed that in eukaryotes, it is the result of a conformational change in ABCE1 that is induced by ATP hydrolysis.

As mentioned previously, peptide release is not a prerequisite for the dissociation of ribosomal subunits or for ABCE1 binding [27]. This is important when ribosome recycling is induced in response to mRNA damage or the assembly of vacant ribosomes as no stop-codon will be detected to initiate termination and peptide release. To overcome this, ABCE1 is able to facilitate peptide release in a similar manner to the eRF1-eRF3-GTP ternary complex, and this has been shown to occur independently of ATP hydrolysis. Here, ATP hydrolysis induces a conformational change in eRF1 that promotes peptidyl-tRNA hydrolysis [26].

Importantly, eRF1 and eRF3 may be sufficient to initiate subunit dissociation; however this is will occur at a slower rate [28].

Mechanisms of recycling in prokaryotes are distinct from those in eukaryotes, with the main difference being the presence of specialized ribosome recycling factor (RRF) that acts together with EF-G to separate the ribosomal subunits in bacteria [4].

Termination of Translation

The next step in the process of translation is termination. In this step an mRNA stop codon indicates that no additional amino acids are to be added to the growing protein. Termination in eukaryotes is facilitated by only two factors (eRF1 and eRF3) and differs significantly to the process in prokaryotes, which involves three factors (RF1, RF2 and RF3) [kcite][19243929][/kcite]. In eukaryotes, two distinct processes must occur for peptide elongation to be successfully terminated; peptide release and the establishment of the post-termination complex. In some cases, where translation must end prior to the detection of a stop codon, the termination step may be skipped and ribosome recycling initiated early. In this case, peptide release will be facilitated by ABCE1 [26].

Termination is triggered by the entry of a stop codon (UAA, UGA or UAG) into the ribosomal A-site. This codon is recognized by a class 1 release factor (RF1). In eukaryotes, this factor (eRF1) binds to the ribosome as part of a pre-assembled ternary complex comprising eRF1, eRF3 and GTP [29]. The stop-codon is recognized by a conserved motifs located at the amino-terminal end of the protein, such as the NIKS motif [30].

eRF1 also assists in peptidyl-tRNA hydrolysis and peptide release from the peptidyl transferase center (PTC). This occurs as a result of GTP hydrolysis by eRF3, which induces a conformational change in eRF1 that allows its Gly-Gly-Gln (GGQ) motif, which is located in the ‘middle’ (M) domain, to enter the ribosomal PTC and facilitate peptidyl-tRNA hydrolysis. This mechanism differs in prokaryotes, where peptide release is required for, and thus precedes, GTP hydrolysis by RF3 [5].

Following GTP hydrolysis and peptide release, the RF3-GDP will dissociate from the protein, leaving behind RF1, which remains bound to the ribosome in what is known as the post-termination complex [26]. This essentially primes the ribosome for ribosomal recycling.

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References

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