3.1 The Translational Apparatus in the Three Domains of Life

Regulation of gene expression at the translational level has received relatively little attention for a long time. Recently, however, especially after the discovery of the small regulator RNAs, miRNA and siRNA, the scientific community has begun to realize that translational regulation is more widespread and important than previously thought, and that it impacts importantly on many essential cell functions.

Translation is known to consist of several distinct steps, initiation, elongation, termination and ribosome recycling. During initiation, the ribosomes, with the aid of a set of proteins termed translation initiation factors, identify the start codon on the mRNA thereby defining the correct reading frame for decoding. This process is rather complex and is fundamental in determining the general rate of translation and the relative abundance of the final protein product.

Translational elongation is itself divided in three steps. The first is adaptation, during which an amino-acylated tRNA enters the ribosomal A site and recognizes the correct codon on the mRNA with the aid of elongation factor 1 (EF1, termed EFTu in Bacteria). The second is trans-peptidation, during which the amino acid carried by the tRNA in the A site is added to the growing peptide chain carried by the tRNA in the P site. The catalytic activity for this reaction is provided by the ribosome itself, specifically by the peptidyl-transferase center of the large ribosomal subunit. The third and final step of elongation, translocation, entails a reciprocal movement of the ribosome and the mRNA, whose final result is a three-nucleotide shift of the mRNA that places the next codon in the A site. Elongation is assisted by elongation factor 2 (EF2, termed EFG in Bacteria).

Termination and ribosome recycling are the final steps of translation, ensuring that the completed polypeptide chain is released from the ribosomes and that the monomeric ribosome is again split into subunits dissociating from the tRNA and the mRNA. As the former ones, this step is also assisted by accessory factors, the termination (or release) factors and the recycling factors.

All of the stages of translation include factors that are G proteins and require therefore the hydrolysis of GTP.

A general scheme of the translation steps and of the factors assisting them in the three domains of cell descent is depicted in Fig. 3.1.

Fig. 3.1
figure 1

Overview of the translation steps and of the factors participating in each of them in the three domains of life. Top: Bacterial translation; middle: Archaeal translation; bottom: Eukaryal translation. The straight line holding the ribosomes represents the mRNA, oriented as illustrated in a 5′-3′ direction. The AUG start codon and one of the possible stop codons (UAA) are shown. The three sets of ribosomes on each mRNA are, from left to right, those engaged in initiation, elongation and termination, respectively. The ribosomal subunits are schematized as divided in two sectors, which represent the P site (on the left) and the A site (on the right). The E site is not shown for simplicity. Only the small ribosomal subunit is shown for the initiation step, since it carries out by itself most of this process. The protein factors participating in each of the steps of translation are shown as spheres close to the ribosomes. The names of the various factors are indicated; their positions relative to the ribosome indicates approximately the main site of interaction. The homologous factors in the different domains are evidenced with the same color; colorless factors are those unique to the domain considered. The question mark for a/eIF6 means that the role of this protein in a specific translation step is still uncertain; therefore, a/eIF6 is shown as a participant in the initiation step or in the termination/recycling step in both Archaea and Eukarya

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Translation may be regulated at any of the above described steps. However, the majority of the regulatory mechanisms act at the level of initiation, influencing the ease with which the ribosomes access the mRNA and/or identify the initiation codon, and thus determining the general rate of decoding. The elongation step may also be subjected to regulation, especially in the case of certain proteins with an idiosyncratic amino acid composition. In the three domains of life, however, translational regulation has attained different levels of accuracy and complexity, and the translational apparatus has diverged accordingly.

To ensure a sophisticated and accurate regulation of protein synthesis, eukaryotic cells have a correspondingly complex translational apparatus. Compared with the bacterial one, the eukaryotic translational apparatus (Fig. 3.1) has a plethora of components, especially as regards the accessory protein factors that assist and modulate the initiation step (Hinnebusch and Lorsch 2012). Recently, it has also emerged that certain eukaryotic cells may synthesize specialized ribosomes, having a slightly altered protein complement, that preferentially translate specific classes of mRNA, incrementing the production of proteins poorly translated by the normal particles (Xue and Barna 2012; Preiss 2016).

Thus, as regards translational regulation, a wide gap appears to exist separating eukaryotic and bacterial cells. The latter have a much simpler translational apparatus, and make use of a minimum of accessory factors for assisting the main target of translational regulation, namely the initiation step. Moreover, transcription and translation are simultaneous events, which restricts the possibility of much sophistication in regulating decoding.

What is true for the Bacteria, however, is not true for the entire prokaryotic world. It has long been known that the Archaea have a translational apparatus that is more complex than the bacterial one and that includes components found in the eukaryotes but not in the bacteria.

The Archaea have ribosomes that are both bigger and richer in proteins than the bacterial ones, even if there is a pronounced variability depending on the archaeal species (Lecompte et al. 2002). Moreover, the Archaea have a set of translation factors decidedly more complex that the bacterial one, especially as regards translation initiation factors (Londei 2005). Some of these factors are specifically shared by the Archaea and the Eukarya, to the exclusion of Bacteria. Finally, the archaeal translational components, from the ribosomal proteins and RNAs to the translational factors, are closer in primary sequence to their eukaryal than to their bacterial counterparts.

The similarity between Archaea and Eukarya regarding the components of the translational apparatus is still puzzling to a large extent, even after over three decades of archaeal studies. Since the Archaea have no nucleus, transcription and translation happen simultaneously as in Bacteria; moreover, the Archaea are known to have polycistronic mRNAs as the Bacteria, implying the ability for the archaeal ribosomes to perform repeated cycles of initiation on the same mRNA.

All this would point to a mechanism of translational regulation generally similar to the bacterial one. Indeed, what little is known about translational regulation in the archaea is in line with this prediction, even if the available data are very scarce.

Yet, the presence of distinct “eukaryotic” features in archaeal translation is undeniable. Some of them have been studied and understood to some extent, while others are still mysterious. In the following, we will try to highlight the similarities between archaeal and eukaryotic translation, taking into account the individual steps of the protein synthesis process.

3.2 Evolutionary Divergence in Translational Initiation

During the initiation step of translation, the ribosomes must identify the correct starting point for decoding on the mRNA, and convey the initiator tRNA on the initiation codon. This apparently simple feat is in actuality tremendously complex, and this is why most of the mechanisms that control speed and efficiency of translation operate at the initiation step.

A staggering amount of research has been performed on eukaryotic as well as on prokaryotic translational initiation. Summarizing in the extreme, the generally accepted model in Eukaryotes is that termed “ribosome scanning”. The small ribosomal subunit (40S), in a complex with several protein factors and with initiator tRNA (met-tRNAi) lands in the vicinity of the capped 5′ end of the mRNA and moves along it until the initiation codon (generally AUG) is found. Then the scanning complex stops, the 60S subunits joins, the initiation factors leave the ribosome and elongation begins (Hinnebusch 2014).

While this model applies to the majority of mRNAs, there are also alternative initiation pathways that take place on uncapped mRNAs. The best known is the internal initiation model, relying upon the presence of special regions on the mRNA to which the ribosome can bind directly (the IRES or ribosome landing pads). Ribosome binding to an IRES may or may not be followed by scanning, but does not require the cap-binding initiation factors (Johnson et al. 2017).

In stark contrast with the complexity of eukaryotic initiation, Bacteria employ an extremely streamlined mechanism, in which the small ribosomal subunit (30S) interacts directly with mRNAs, often polycistronic, through the so-called TIR (Translation Initiation Region). This includes the initiation codon preceded, in many but not in all cases, by the Shine-Dalgarno sequence which specifically pairs with the 3′end of the 16S rRNA. In this process just three initiation factors are sufficient for the identification of the start codon and for correctly positioning the ribosome on it.

Interestingly, of the three bacterial initiation factors, two are universally conserved proteins, found in all three domains of life. However, one of these, the factor called IF2, functions as the tRNAi binding factor, a role that is not conserved in either Archaea or eukaryotes (Gualerzi and Pon 2015).

The Archaea have many apparent similarities with the Bacteria, such as being endowed with prokaryotic-sized ribosomes (70S). Their mRNAs also share common characteristics with those of Bacteria, in fact, they are often polycistronic and may contain Shine-Dalgarno sequences, albeit these are infrequent in certain archaeal species (Benelli et al. 2016). Archaeal mRNAs also hold unique features such as, in many cases, the lack of a 5′UTR (leaderless mRNAs). Leaderless mRNAs are unevenly distributed among Archaea: they are the majority of mRNAs in certain species of the phylum Crenarcheota, while being much less frequent in Euryarcheota such as methanogens.

However, with respect to the Bacteria, the Archaea have an enlarged set of translation initiation factors, although it is unclear why it should be so. To date, the recognized translation initiation factors in Archaea are the proteins termed aIF2, aIF1, aIF1A, aIF5B. Another factor, aIF6, is certainly involved in translation but its function is still uncertain. Two of these five proteins, aIF1A and aIF5B, are also found in all Bacteria. They are, respectively, homologous to the bacterial factors IF1 and IF2. The factor termed IF1 (or SUI1) in Archaea and Eukarya is also present in some, but not all, bacterial phyla, being sometimes termed YCiH. The remaining two proteins, a/eIF2 and a/eIF6, are shared exclusively by the Archaea and the Eukarya, and presumably the latter have inherited them from their archaeal ancestor (Benelli et al. 2016).

The Eukarya, of course, have many more initiation factors that are not found in either of the prokaryotic domains. Among these, the factors that interact with the cap at the mRNA 5′ and that guide the 40S subunits during scanning. An overview of the translation initiation factors in the three domains of life is presented in Table 3.1.

Table 3.1 Translation initiation factors in the three domains of life
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Clearly, the most interesting question is why the Archaea should have a translation initiation apparatus more complex than the bacterial one, and particularly why they should share with the eukaryotes a specific set of factors. While the answer to this question still remains elusive, the progress of our knowledge on archaeal initiation has begun to elucidate the similarities and differences existing in the features of translation initiation that employ factors specifically shared by the bacteria and the archaea.

3.2.1 The a/eIF1/SUI1 Factors

The protein termed a/eIF1 or SUI1 is one of the translation initiation factors, universally shared by the Archaea and the Eukarya, but lacking in most Bacteria. To be sure, genes encoding homologues of a/eIF1 have been found in certain bacterial phyla, such as the proteobacteria and the cyanobacteria, but are apparently missing in all other species (Kyrpides and Woese 1998). Studies in E. coli have established that the SUI1 homologue is not essential (Baba et al. 2006) and that it probably does not participate in translational initiation, although it may be involved in the expression of certain stress-related genes (Osterman et al. 2015).

The peculiar evolutionary distribution of a/eIF1/SUI1 is compatible with the idea that this gene was originally present in the common ancestor of all cell domains, but was subsequently lost by the Bacteria, probably because it was replaced by another, bacterial specific factor, better adapted to perform its appointed function.

But what is the function of a/eIF1/SUI1? In Eukaryotes, where it has been studied extensively, eIF1 is known to have important roles in translational initiation. It binds to the 40S subunits and prevents the premature joining of the 60S particle. Also, and more importantly, it discriminates against non-canonical initiation codons, helping to ensure the fidelity of translational initiation. Moreover, eIF1 is essential for the process of ribosome scanning, whereby the 43S initiation complex, consisting of the 40S ribosomal subunit and of several initiation factors (including IF1 and eIF1A, another universally conserved protein) moves along the mRNA to locate the translation start codon. Cryoelectron microscopy studies suggested that eIF1 and eIF1A maintain the initiating ribosome in an “open”, scanning-competent, conformation until the start codon is located, and the first codon/anti-codon base-pairing has been established (Passmore et al. 2007). Then the complex undergoes a conformational change and eIF1 is released (Maag et al. 2005; Cheung et al. 2007).

The release of eIF1 is believed to free the C terminus of eIF1A for interactions with eIF5, which stabilizes the closed state of the complex (Maag et al. 2006).

In Archaea, the function of aIF1 has been studied to some extent in the extreme thermophile Sulfolobus solfataricus. It has been established that, as in the Eukaryotes, the factor binds specifically to the 30S ribosomal subunits and is not found on elongating 70S ribosomes, arguing for a specific role in translation initiation (Hasenöhrl et al. 2006).

The binding site of aIF1 on the 30S subunits has also been defined, and found to coincide with that occupied by the corresponding eukaryotic factor on the 40S subunit. Experiments of hydroxy-radical probing have identified helices 23 and 24 of the 16S RNA as the region protected by aIF1 binding, a region that corresponds with that protected by eIF1 on the 40S subunits (Hasenöhrl et al. 2009).

The function of the archaeal factor also apparently corresponds to that of its eukaryal counterpart, specifically regarding the role in determining the fidelity of initiation codon choice. Indeed, aIF1 discriminates against ribosome binding to a mRNA having the non-canonical initiation codon AUU (Hasenöhrl et al. 2009). It is interesting to note that this important “fidelity function” also exists in Bacteria, but it is performed by the bacterial-specific factor IF3, that has no evident homology with a/eIF1. As said above, IF3 probably has replaced IF1/SUI1 in the course of bacterial evolution. The reason for discarding a universal factor for a new one is not evident, but it is probably due to the progressive streamlining of the translation initiation mechanism (and in general, of the gene expression process) that took place once the bacterial lineage separated from the common stem of the tree of life. The lack of a comparable streamlining in Archaea is conceivably due to the fact that the Archaea mostly occupy “extreme” ecological niches where competition for fast growing is not so hard as in the bacterial world.

3.2.2 The a/eIF2 and IF2/IF5B Factors

a/eIF2 is a trimeric protein specifically shared by the Archaea and the Eukarya but lacking in Bacteria. Although the Bacteria do possess a translation initiation factor termed IF2, this is not homologous to the same-named archaeal/bacterial protein but to the factor termed IF5B in the other two domains. Therefore, IF2/5B is a universal factor, while a/eIF2 is specific of the archaeal and eukaryal domains only (Kyrpides and Woese 1998) (Table 3.1).

The terminology of these proteins is already confusing, but the confusion is even greater when it comes to their function. Regarding IF2/IF5B, since this protein is one of the two universally conserved initiation factor, one would expect a correspondingly universal and presumably essential function. But it is not so. In Bacteria, IF2 is a truly central player in translational initiation: it interacts with the initiator tRNA (fmet-tRNAi) and promotes its accommodation in the ribosomal P site, at the same time favoring subunit joining (Gualerzi and Pon 2015). By contrast, in Archaea and Eukarya, the initiator tRNA (met-tRNAi) binding factor is the trimeric IF2 (Pedullà et al. 2005; Schmitt et al. 2010), that, as said above, has no counterpart in Bacteria. The archaeal/eukaryal homologue of bacterial IF2, a/eIF5B, does not bind met-tRNAi but apparently still promotes subunit joining, also stabilizing the interaction of met-tRNAi in the P site (Maone et al. 2007).

Therefore, when it comes to the fundamental task of recognizing the specific initiator tRNA and promoting its interaction with the ribosomal P site, there seems to be a clear-cut evolutionary divergence separating the bacterial domain from the archaeal and eukaryal ones.

In the Archaea, moreover, the trimeric IF2 has a peculiar and unexpected function. It interacts specifically with the tri-phosphate 5′ end of the mRNA protecting it against 5′-end degradation Such interaction takes place both with the trimeric form of aIF2 and with its individual subunit γ and is favored when the factor is in a cytoplasmic, free state (Hasenöhrl et al. 2008). Instead, when aIF2 is in a ribosome-bound state, it has a much stronger affinity for met-tRNAi. This dual function of aIF2 is thought to prevent mRNA degradation under unfavorable nutritional conditions, when ribosome synthesis temporarily stops and ribosomes become fewer (Hasenöhrl et al. 2008).

These observations have led to speculate that, in Archaea, IF2 evolved originally to protect the mRNAs against 5′-end degradation, thus prefiguring a sort of cap-binding protein system reminiscent of that seen in modern eukaryotic cells. Archaeal mRNAs have no real “caps”, but their free 5′ tri-phosphate end, interacting specifically with aIF2, would perform the same protective function as the eukaryotic cap. However, the “capping” system seen in modern eukaryotic cells must have evolved de novo during the separate evolution of eukaryal translation, since it is based on components that are specific of the Eukarya and have no counterparts in the other cell domains.

A possible evolutionary history of the tRNAi binding proteins in the three domains of life has been recently described in detail elsewhere (Benelli et al. 2016).

3.2.3 The a/eIF6 Factors

In both Archaea and Eukarya, the translation factor IF6 is a small (27 kDa), monomeric protein that binds specifically to the large ribosomal subunit. The role initially proposed for this factor in eukaryotes was that of preventing the association of the 40S and 60S subunits until the pre-initiation complex was correctly positioned on the start codon (Valenzuela et al. 1982). However, it was later observed that eIF6 is located also in the nucleolus and that its loss affects the biogenesis of 60S particles, suggesting that the protein has an important role in ribosome biosynthesis (Si and Maitra 1999).

aIF6, the archaeal homologue, is a few amino acids shorter than its eukaryal counterpart, but shares otherwise a high degree of homology with it. Its three-dimensional structure has been solved (Groft et al. 2000). It shows a peculiar fold, termed “pentein” because it is composed by a repetition of five very similar domains. The structure of the eukaryal counterpart, modelled on the basis of the archaeal one, is essentially the same.

a/e IF6 binds with high affinity to the large ribosomal subunit, either 50S or 60S. The binding site, first determined for the archaeal factor (Benelli et al. 2009; Greber et al. 2012) and later also for the eukaryal one (Klinge et al. 2011), lies on the surface of the large subunit that interacts with the small subunits, thus justifying its role as an anti-association factor. This region of the ribosome is rather protein-poor; however, IF6 is located in the vicinity of L14p and L24e, and, in Archaea at least, interacts with the former (Benelli et al. 2009).

To date, the role in translation of IF6 remains puzzling. In both the Archaea and the Eukarya, only about 1 in 10 large ribosomal subunits carry a/eIF6 in the cytoplasm. Moreover, the interaction of a/eIF6 with the ribosome is quite strong, and specific factors are required for its release. As regards eukaryotic ribosomes, two different mechanisms have been proposed for eIF6 release. One posits that eIF6 detachment from the 60S subunits is promoted by the GTPase, Efl1, which acts in concert with the ribosome-binding factor Sdo1 (also called SBDS) to couple GTP hydrolysis with IF6 release (Weis et al. 2015). Another proposed mechanism suggests that eIF6 release is triggered by the phosphorylation of the factor, in turn promoted by translation-stimulating signalling transduced by the ribosome-bound kinase RACK1 (Ceci et al. 2003). It is unclear whether these mechanisms co-exist or operate in different circumstances or in different cells.

In eukaryotes, the current consensus model for eIF6 function has it that the factor intervenes in the final maturation steps of the large ribosomal subunit. Immature 60S ribosomes would be shipped to the cytoplasm carrying bound eIF6. The release of the factor, by whichever mechanism, would allow the particles to participate in translation. In this model, the main role of eIF6 would be that of fine-tuning translation by regulating the amount of available 60S subunits.

Compared to Eukarya, much fewer data are available on the function of archaeal IF6. It is known that aIF6 binds tightly and specifically to the 50S ribosomal subunit and thereby inhibits subunit association (Benelli et al. 2009). 50S subunits carrying aIF6 are unable to participate in translation, since they are not found in either 80S ribosomes or in polysomes. However, the mechanism for aIF6 release from the 50S subunit is still unknown. Phosphorylation is in all probability not involved, since efforts to determine whether aIF6 undergoes this type of modification have been unsuccessful (Benelli and Londei, unpublished work). However, the Archaea do harbour a homologue of eukaryal Sdo1/SBDS, which closely corresponds to its eukaryal counterpart in sequence and structure.

The function of aSdo1/SBDS is currently under scrutiny in our laboratories. Preliminary experiments performed with the thermophilic archaeon S. solfataricus seem to indicate that addition of recombinant aSdo1 to ribosomes or cell lysates promotes the release of aIF6 in a GTP-dependent manner. Moreover, aSdo1 appears to bind stoichiometrically to the 50S subunit (Benelli, La Teana and Londei, unpublished work). However, the Archaea do not have any evident homologue of the Efl1 protein, suggesting that another, archaeal-specific, GTPase must be involved in the process. Experiments currently under way in our laboratory are aimed at identifying such a GTPase, and at elucidating the mechanism promoting aIF6 release from archaeal large subunits.

As regards the function of archaeal aIF6, there is very little solid evidence so far. Undoubtedly, the protein prevents subunit association and inhibits protein synthesis when added in excess to cell lysates (Benelli et al. 2009), but the physiological significance of this remains elusive. A later study suggests that the main role of aIF6 might be that of promoting ribosome recycling, stimulating the dissociation of 70S ribosomes at the end of each translation cycle (Barthelme et al. 2011). Further data are, however, needed to confirm this surmise. Finally, as suggested for the eukaryotic homologue, aIF6 might participate in ribosome biosynthesis, but the issue remains entirely to be explored experimentally.

The confusion about the role in translation of a/eIF6 is all the more frustrating since this factor, in eukaryotes at least, has clearly a very important role in regulating certain crucial cellular processes. Remarkably, eIF6 over-expression is observed in many natural cancers, while, conversely, eIF6 haplo-insufficiency protects against certain types of tumours (Gandin et al. 2008). Moreover, the over-expression of eIF6 has been described to produce developmental defects in Xenopus (De Marco et al. 2010, 2011).

Unfortunately, there are no data in Archaea to show whether aIF6 imbalances have any kind of physiological effects. It is only known that aIF6 is over-expressed under stress conditions, a fact that may suggest a role in controlling cellular behaviour roughly similar to that observed for its eukaryotic counterpart. However, we are still a long way from understanding all the functional facets of this fascinating factor, let alone the motive for its evolutionary conservation in the archaeal/eukaryal line. It is to be hoped that a better understanding of the function of aIF6, the evolutionary forerunner of eIF6, will also help in elucidating the function of the latter.

3.3 Elongation

Elongation is the most conserved among the steps of protein synthesis. In all organisms, the elongation cycle entails the participation of two accessory factors. The first of these, called EF1 in Eukarya and Archaea and EFTu in bacteria, accompanies aminoacyl-tRNA in the ribosomal A site and controls the correctness of codon-anticodon interaction. The second, called EF2 in Archaea and in Eukarya, and EFG in Bacteria, assists the process of translocation, i.e. the movement of the ribosome one codon further down the mRNA. Both elongation factors are G proteins that hydrolyze GTP as an essential part of their function. The prokaryotic proteins are somewhat smaller than the eukaryotic ones, but they are clearly homologous and their mechanism of action is strictly conserved throughout evolution.

Recent research, however, has unveiled certain specialized aspects of elongation that have received scanty attention until now. They regard the function of another evolutionarily conserved translation factor, the protein called EFP in bacteria and IF5A in Archaea and Eukarya. The existence of this protein has been known for a long time, but the details of its function have only recently been analyzed. It is interesting to review the relevant data, since this factor may have many functional facets which we are only just beginning to understand.

The eukaryotic and the bacterial proteins were discovered in the ’70s. Their different names are due to the fact that initially the eukaryal protein was classified as an initiation factor (then named eIF4D) and the bacterial one as an elongation factor. In spite of this, both proteins were characterized as having the same activity: the ability to stimulate the formation of (f)Met-Puromycin in vitro (Benne et al. 1978; Glick and Ganoza 1975).

The eukaryal protein was found to contain a unique post- translational modification: hypusination. This modification is carried out in two successive enzymatic reactions. In the first deoxyhypusine synthase (DHS) transfers the aminobutyl moiety of spermidine to the ε-amino group of a specific lysine located in the N-terminal domain of the protein, in the second reaction the intermediate, deoxyhypusine, is transformed into hypusine by deoxyhypusine hydroxylase (DOHH) (Cooper et al. 1983; Park et al. 2010).

More recently, a post-translational modification, lysinylation, has been identified also in the bacterial protein. This modification occurs in three steps catalyzed by the following enzymes: YjeK, which converts a free S-α-Lys to R-β-Lys, YjeA, a paralog of lysyl-tRNA synthetases that transfers the R- β-Lys to the ε-amino group of a specific lysine and, finally, YfcM which hydroxylates the lysysl-lysine residue (Reviewed in Rossi et al. 2014).

3D structures are available from Bacteria, Archaea, Protozoa, yeast and human, and they show overall a similar organization: the bacterial protein folds into three domains while in all other cases the proteins are organized in two domains whose structure is superimposable with the first two bacterial domains (Fig. 3.2). The basic N-terminal domain contains the site of post-translational modification in an exposed loop while the acidic C-terminal domain is characterized by an OB-fold (Reviewed in Dever et al. 2014).

Fig. 3.2
figure 2

The three-dimensional structures of archaeal (Pyrococcus horikoshii, PDB: 1IZ6) and eukaryal (Saccharomyces cerevisiae, PDB: 3ER0) IF5A compared with their bacterial homologue (Thermus thermophilus, PDB: 1UEB) EF-P. The protein regions with a β-sheet conformation are depicted in yellow, the α-helices are purple. The arrows point at the sites of modification: hypusination for aIF5A and eIF5A and lysinylation for EF-P

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A complete functional characterization has been obtained, for both proteins, only in more recent years. Studies have established a role for both eIF5A and EF-P in translation elongation more than in initiation: they are able to promote the synthesis of proteins containing successive residues of proline (PPP or PPG) (Gutierrez et al. 2013; Ude et al. 2013; Doerfel et al. 2013). Synthesis of these type of sequences, in fact, causes the ribosome to stall, and only the intervention of eIF5A/EF-P guarantees the recovery of the elongation process.

According to the model suggested by structural studies (Schmidt et al. 2016; Melnikov et al. 2016), the factor would bind to stalled ribosomes, trapped in a pre-translocational state and with a free E-site, and interact with A76 residue of a P-site tRNA, via its hypusine moiety. This interaction stabilizes the tRNA facilitating in this way peptide bond formation.

In addition to its direct role as a translation factor, eIF5A has been related to a variety of cellular processes including: mRNA decay (Zuk and Jacobson 1998), cell cycle progression (Hanauske-Abel et al. 1994), apoptosis (Caraglia et al. 2003), cell polarity (Chatterjee et al. 2006; Zanelli and Valentini 2005), retroviral infection (Hoque et al. 2009) and stress responses (Gosslau et al. 2009). Whether eIF5A is endowed with different functions or if this pleiotropic behavior results from secondary effects of its role as a translation factor, remains to be clarified.

The eukaryal and the bacterial proteins, as described above, have been extensively characterized while knowledge on the archaeal one is still very limited.

The presence of a hypusinated protein in Archaea was discovered several years ago: the protein was purified from Sulfolobus acidocaldarius DSM 639, it is a protein of 135 AA with a mass of about 15 KDa and pI of 7.8, mainly present in the post-ribosomal fraction (Bartig et al. 1992).

All archaea analyzed so far contain aIF5A, but some have a hypusinated factor, while some others contain the deoxyhypusinated form and very few, both versions of the protein (Bartig et al. 1990). Despite the presence of the different forms of the modified protein, the mechanism of hypusination remains a mystery since so far only the first enzyme involved in aIF5A modification, the DHS enzyme, has been identified in archaeal genomes while no homologs of the second enzyme, DOHH, seem to be present.

A recent paper has shed some light on the Haloferax volcanii modification pathway (Prunetti et al. 2016); this organism contains exclusively the deoxyhypusinylated version of aIF5A, spermidine is absent while agmatine and cadaverine represent the main polyamines present. The authors, therefore, propose a model of deoxyhypusine synthesis in H. volcanii that differs substantially from the canonical eukaryotic pathway: in the first reaction, the DHS enzyme transfers agmatine to the aIF5A lysine while in the second reaction the agmatinase enzyme leads to production of deoxyhypusine.

The situation in other Archaea might be similar with the involvement of enzymes completely unrelated to the eukaryal DOHH; in alternative the two modification reactions could be catalyzed by a single enzyme, DHS, endowed with a bifunctional activity. The latter possibility is supported by the recent discovery and characterization of such an enzyme in T. vaginalis (Quintas-Granados et al. 2016).

In any case modification of the lysine seems to be important since at least some archaea (S. acidocaldarius) are sensitive to the DHS inhibitor GC7, which causes a rapid and reversible arrest of growth (Jansson et al. 2000).

The aif5a gene appears to be essential at least in H. volcanii (Gäbel et al. 2013) and in S. acidocaldarius (La Teana, Londei and Albers, unpublished results). Its participation in the translation process has been inferred on the basis of its homology to the other factors but has not yet been demonstrated.

Experiments carried out in our laboratories and aimed at clarifying its role have confirmed that in S. solfataricus cell lysates aIF5A is mainly present in the post-ribosomal supernatant. However, when the lysates are programmed for translation by addition of an exogenous mRNA and fractionated on sucrose density gradients, hypusinated aIF5A becomes localized on 70 ribosomes, suggesting a conserved role in translation.

The hypothesis of a participation in the translation process strictly linked to the rescue of proteins containing polyproline motives, however, needs to be verified.

A genome-wide analysis has shown that these proteins are not so common in both Bacteria and Archaea but their abundance increases with the complexity of the organism going from Prokaryotes to Eukaryotes (Mandal et al. 2014). The frequencies of proteins containing PPP and PPG motifs range from 2.0 to 2.5% in Bacteria and Archaea to more than 20% in H. sapiens.

On the other hand, polyproline might not be the only motif whose translation is dependent on this factor, as it has been reported for EF-P in some bacterial species (Hersch et al. 2013).

As mentioned above, eIF5A could be involved in processes other than translation; in particular, several reports have characterized it as an RNA binding protein. The homology between the eukaryal and the archaeal protein suggests that this activity might be shared also by aIF5A and the finding that in some species of Halobacterium the protein shows an RNA binding and degrading activity is in agreement with this hypothesis (Wagner and Klug 2007).

The most interesting aspect about this elongation factor is the conservation of its modification. Both proteins, EF-P and a/eIF5A, are targets of unique modifications, and the most conserved regions with the highest sequence homology are located around the site of modification. The modifications nevertheless are different, β-lysinylation and hypusination, and are catalyzed by completely unrelated enzymes, which are themselves highly conserved within each domain.

As said above, one hypothesis is that these factors and their enzymes have co-evolved to guarantee the synthesis of some essential protein containing proline-rich sequences. Starosta et al. (2014) have analyzed the number and conservation of polyproline-containing proteins across 1273 bacterial, 205 archaeal and 98 eukaryotic genomes finding one proline triplet which is invariant in the Valyl-tRNA synthetase (ValS) genes from all organisms. It may be that this essential protein sufficed by itself to induce the evolution of a factor specifically devoted to stimulate its synthesis. However, it is also possible that in prokaryotes EFP/aIF5A plays some other important role in addition to promoting the translation of poly-pro containing proteins. A fuller investigation of the function of archaeal aIF5A should help to answer this question.

3.4 Conclusions

Despite the considerable advances in our knowledge in the last decade or two, the evolutionary history of the translation process remains to be written in many essential aspects. The unexpected complexity detected in the Archaea regarding some features of translation, and the general closeness between Archaea and Eukarya in the sequences of many translational components, further confirm the now generally accepted idea that Archaea and Eukarya are closely related in evolution.

The prevalent view of the general evolutionary tree of life, that envisages Bacteria as the most antique branch thereof, with Archaea and Eukarya sharing a common evolutionary path before separating in their turn, is in theory open to two different interpretations. The common ancestor of all cells might have had a translational apparatus with a minimal set of components (small, protein poor ribosomes, the two universally conserved initiation factors IF2/IF5B and IF1/IF1A, two elongation factors). After the separation of the bacterial domain, other components could have been added during the common evolution of Archaea and Eukarya, and still others during the separate evolution of the Eukarya.

Alternatively, the last common ancestor of the three domains of life might have had a translation apparatus very similar to that of thermophilic Crenarchaea, deemed to be the oldest branch of the archaeal tree: relatively larger and protein-richer ribosomes and an enlarged set of translation initiation factors, including a/eIF2 (or at least its gamma-subunit), IF1/SUI1 and perhaps a/eIF6. The possible presence of a/eIF2, in either a trimeric or monomeric form, in the common ancestor, is also suggested by the fact that the majority of crenarcheal mRNAs are leaderless, i.e. lack entirely or almost so a 5′ untranslated region. It has long been known (Grill et al. 2000) that leaderless mRNAs are universally translatable by the ribosomes of all extant cells, and that therefore are the likely ancestral form of genetic message. If most ancestral mRNAs had no 5′ leader, it might have been important to protect their 5′ termini until they could be translated.

Under the above scenario, the Bacteria lost some translational components during their separate evolution, remodeling others to perform new functions. This would be, for instance, the case of IF2/5B, which would have acquired the ability of interacting with the initiator tRNA (formerly performed by the lost a/eIF2 or by its gamma subunit, see Benelli et al. 2016), retaining at the same time the ability of promoting subunit joining. IF1/SUI1 is another likely case of a factor lost in the bacterial line, replaced by the new entry IF3. The presence of the IF1/SUI1 gene, most probably in an inactive form, in certain bacterial phyla, might be considered a relic of this loss-replacement process.