Unique modifications of translation elongation ... - Wiley Online Library

REVIEW ARTICLE

Unique modifications of translation elongation factors Eva Greganova*, Michael Altmann and Peter Bu¨tikofer Institute for Biochemistry and Molecular Medicine, University of Berne, Switzerland

Keywords diphthamide; eEF1A; eEF2; eIF5A; ethanolamine phosphoglycerol; hypusine; protein modifcation; translation elongation Correspondence P. Bu¨tikofer, Institute of Biochemistry and Molecular Medicine, University of Bern, Bu¨hlstrasse 28, 3012 Bern, Switzerland Fax: +41 31 631 3737 Tel: +41 31 631 4113 E-mail: [email protected] M. Altmann, Institute of Biochemistry and Molecular Medicine, University of Bern, Bu¨hlstrasse 28, 3012 Bern, Switzerland Fax: +41 31 631 3737 Tel: +41 31 631 4127 E-mail: [email protected]

Covalent modifications of proteins often modulate their biological functions or change their subcellular location. Among the many known protein modifications, three are exceptional in that they only occur on single proteins: ethanolamine phosphoglycerol, diphthamide and hypusine. Remarkably, the corresponding proteins carrying these modifications, elongation factor 1A, elongation factor 2 and initiation factor 5A, are all involved in elongation steps of translation. For diphthamide and, in part, hypusine, functional essentiality has been demonstrated, whereas no functional role has been reported so far for ethanolamine phosphoglycerol. We review the biosynthesis, attachment and physiological roles of these unique protein modifications and discuss common and separate features of the target proteins, which represent essential proteins in all organisms.

*Present address Swiss Tropical and Public Health Institute Socinstrasse 57, 4002 Basel, Switzerland (Received 7 April 2011, revised 12 May 2011, accepted 26 May 2011) doi:10.1111/j.1742-4658.2011.08199.x

Introduction Several hundred protein modifications are known today, making proteomes far more complex than could be predicted by the encoding genomes. Covalent modifications modulate the biological functions or change the subcellular location of proteins and affect interactions of proteins with a variety of molecules, such as nucleic acids, lipids or other proteins [1–3]. Particular modifications are usually present on many proteins

and often proteins carry several modifications at multiple amino acid residues [4]. The synthesis and attachment of protein modifications often involves multiple gene products and sets of metabolites, making these events costly for a cell in terms of substrate and energy requirements. On the other hand, modifications may generate additional functions for proteins or allow novel pathways of regulation, providing a cell with

Abbreviations DHS, deoxyhypusine synthase; DOOH, deoxyhypusine hydroxylase; e(a)EF1A, eukaryotic (archaeal) elongation factor 1A; e(a)EF2, eukaryotic (archaeal) elongation factor 2; e(a)IF5A, eukaryotic (archaeal) initiation factor 5A; EF-G, bacterial elongation factor 2; EF-P, bacterial elongation factor P; EF-Tu, bacterial elongation factor 1A; EPG, ethanolamine phosphoglycerol; PE, phosphatidylethanolamine.

FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS

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extra means to diversify and develop. While some modifications are transient and thus depend on rapid attachment and removal of molecules from target proteins, others are stable and attached to proteins shortly after their synthesis or before degradation [4]. Among many protein modifications, three are exceptional in that they only occur on single proteins: ethanolamine phosphoglycerol (EPG), diphthamide and hypusine. Remarkably, the corresponding proteins carrying these modifications, eukaryotic elongation factor 1A (eEF1A), eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 5A (eIF5A) respectively, are all involved in the elongation steps of translation. Elongation of polypeptide chains during translation is a conserved process among prokaryotes and eukaryotes. Single steps of elongation consist of (a) binding of aminoacyl-tRNAs to the A(minoacyl)-site of the ribosome, (b) peptide bond formation with the adjacent peptide-tRNA at the P(eptidyl)-site and (c) translocation of the extended peptide-tRNA from the A-site to the P-site and of the previously loaded tRNA from the P-site to the E(xit)-site. These steps are well conserved between organisms and the enzymatic involvement of ribosomal RNA at the transpeptidation center is nowadays generally accepted. Accordingly, homologs of most factors involved in elongation can be found across bacterial, archaeal and eukaryotic genomes. eEF1A, eEF2 and eIF5A are phylogenetically among the most highly conserved proteins. Their biological roles during elongation of translation are as follows: eEF1A (called EF-Tu in bacteria and aEF1A in archaea), one of the most abundant cytosolic proteins, catalyzes binding of aminoacyl-tRNAs to the A-site of the ribosome. In addition, it has been reported to participate in a variety of other functions (so called moonlighting functions; see below). In contrast, eEF2 (called EF-G in bacteria and aEF2 in archaea) is involved in translocation of the peptide-tRNA complex from the A- to the P-site, while eIF5A (called EF-P in bacteria and aIF5A in archaea) directly stimulates protein elongation, yet its precise mode of action on the ribosome is unclear [5]. Bacterial EF-P facilitates the proper positioning of the initiator-tRNA-methionine complex at the P-site [6]. Both eEF1A and eEF2 are GTP-binding proteins, i.e. their enzymatic activity requires the hydrolysis of GTP to GDP. Interestingly, GTPases involved in translation elongation show a remarkable structural similarity pointing at a common ancestral GTPase (reviewed by [7]). Its presumed function was to transport aminoacyl-tRNAs to an ancestral membranebound self-folding RNA, which catalyzed peptide bond 2614

formation and constituted the original peptidyltransferase center that evolved later into the corresponding domain of the ribosomal large subunit. Co-evolution of translational GTPases with ribosomal structures may have occurred to allow interaction of GTPases with ribosomal structures by addition of new structural elements [7]. In accordance with the concept of co-evolution between proteins and RNA structures, elongation (and termination) factors of translation show a remarkable molecular mimicry between proteins and tRNAs. For example, the crystal structure of EF-G from Thermus thermophilus perfectly fits the structure of the ternary prokaryotic EF-Tu-GDPNP-PhetRNAPhe complex [8]. In addition, the crystal structure of EF-P from Escherichia coli with its post-translational lysine modification resembling the covalently bound amino acid lysine charged to the 3¢ end of a tRNA (see below) mimics the structure of a charged tRNA [9]. The unique modifications attached to eEF1A, eEF2 and eIF5A have been known for decades. In addition, their biosynthetic precursors and pathways for production and attachment to protein have been partially established (see below). Surprisingly, their biological functions have remained elusive despite the fact that EPG, diphthamide and hypusine are attached to essential proteins involved in a highly conserved process, i.e. elongation of protein translation, and that speciesspecific variants of the three proteins have been crystallized and their 3D structures solved. The aim of this review is to describe common and separate features of EPG, diphthamide and hypusine attachment to their respective acceptor proteins. Interestingly, despite the fact that not only the function but also the 3D structures of e(a)EF1A ⁄ EF-Tu, e(a)EF2 ⁄ EF-G and e(a)IF5A ⁄ EF-P proteins have been conserved during evolution (Fig. 1), the presence of EPG, diphthamide and hypusine shows striking differences: whereas hypusine (or lysine) attachment to e(a)IF5A ⁄ EF-P proteins has been demonstrated in all three domains of life, diphthamide modification has only been found in e(a)EF2 of eukarya and archaea but not in EF-G of bacteria, while EPG has so far only been reported in eEF1A of eukarya (Fig. 1).

Eukaryotic elongation factors and their unique modifications eEF1A and EPG eEF1A represents an essential protein involved in peptide chain elongation in all eukaryotic cells. It interacts in its GTP-bound form with an aminoacylated tRNA


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Fig. 1. 3D structure of translation elongation factors. The 3D structure of representative examples of e(a)IF5A ⁄ EF-P (top row), e(a)EF2 ⁄ EF-G (middle row) and e(a)EF1A ⁄ EF-Tu (bottom row) proteins is drawn to demonstrate the structural similarity between eukarya, archaea and bacteria. The position of the unique modifications hypusine (Hyp), diphthamide (Dph) and ethanolamine phosphoglycerol (EPG) attached to conserved amino acids (numbered) is indicated by arrows. Structures represent eIF5A from Homo sapiens (UniProt, Q6IS14), aIF5A from Sulfolobus acidocaldarius (GenBank, CAA44842) and EF-P from E. coli (GenBank, AP_004648), eEF2 from S. cerevisiae (UniProt, P32324), aEF2 from H. salinarum (UniProt, Q9HM85) and EF-G from T. thermophilus (UniProt, Q5SHN5), and eEF1A from Mus musculus (GenBank NP_034236), aEF1A from H. salinarum (GenBank, NP_281202) and EF-Tu from E. coli (GenBank, YP_001465471), and are drawn using the PYMOL program [99].

to mediate binding to the acceptor site of a ribosome via codon–anticodon interaction. Following ribosomedependent hydrolysis of GTP, eEF1A dissociates from the ribosome in its GDP-bound form and interacts with nucleotide exchange factor eEF1B (called EF-Ts in bacteria) that replaces GDP by GTP to reactivate eEF1A (reviewed in [10,11]). Crystal structures of eEF1A in complex with subunits of eEF1B show that eEF1A from Saccharomyces cerevisiae consists of three distinct structural domains [12,13]. The N-terminal

domain I contains the binding site for guanine nucleotides whereas binding of aminoacyl-tRNAs occurs in domain II [12,14–17]. In addition, domains I and II share the recognition site for the a-subunit of eEF1B [12,13]. In S. cerevisiae, domain III has been shown to harbor the binding site for the fungal-specific elongation factor 3 [18,19]. Beside its canonical role in protein synthesis, eEF1A has been shown to also bind to cytoskeletal proteins and mediate their interactions [20–22]. This function, which has been localized to


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domains II and III, seems not to be connected to its role during polypeptide elongation [21,22]. In addition, eEF1A was reported to be involved in signal transduction processes [23], nuclear export of proteins [24] and import of tRNAs into mitochondria [25]. Based on the high conservation of the primary sequence of eEF1A among eukaryotes (Fig. S1) and its highly conserved role during protein synthesis, it can be speculated that many interactions with its binding partners are conserved among other eukaryotic organisms. The activity of eEF1A during peptide synthesis has been reported to be modulated by post-translational modifications such as phosphorylation [26,27], lysine methylation (reviewed in [28,29]) and C-terminal methyl-esterification [30]. The precise role of these modifications is unclear (reviewed in [31]). In contrast, no studies have been reported on the role of EPG that is attached to conserved glutamate residues in eEF1A of several eukaryotes (Fig. S1). Chemical and mass spectrometric analyses demonstrated that murine [32], rabbit [33] and carrot [34] eEF1A contain two EPG modification sites, located in domains II and III. In contrast, although both glutamates are conserved in eEF1A of the protozoan parasite Trypanosoma brucei (Fig. S1), trypanosome eEF1A is modified only by a single EPG moiety attached to Glu362 in domain III [35] (Fig. 2A). Amino acid point mutations of the modification site in T. brucei eEF1A were found to prevent attachment of EPG, even when glutamate was

replaced by aspartate [36], demonstrating that EPG attachment is strictly specific for glutamate. Interestingly, S. cerevisiae represents the only eukaryote so far reported where eEF1A is not modified with EPG [28], although the glutamate residue in domain III is conserved among yeast and other eukaryotes (Fig. S1). Amino acid sequence comparisons between eEF1A and EF-Tu show that eukaryotic EPG modification sites are not strictly conserved in bacteria (Figs S2 and S3). For E. coli, the lack of EPG modification has been proven experimentally [32]. Recent analyses of aEF1A from Halobacterium salinarum and Haloquadratum walsbyi showed no evidence for the presence of EPG (E. Greganova, R. Vitale, A. Corcelli, M. Heller & P. Bu¨tikofer, unpublished results) suggesting that EPG is absent in archaea. Interestingly, despite the high amino acid sequence identity between eEF1A proteins from different eukaryotes, the residues around the EPG modification sites are less well conserved (Fig. S1) suggesting that they may not be essential for EPG attachment [36]. Additionally, when expressing eEF1A deletion mutants or chimeric proteins consisting of domain III of T. brucei eEF1A fused to soluble reporter proteins, a peptide consisting of 80 amino acids of domain III of eEF1A was found to be sufficient for EPG attachment to occur, indicating that EPG attachment is dependent on the three-dimensional structure of domain III rather than the sequence of amino acids around the attachment site [36].

Fig. 2. Attachment of EPG to eEF1A. (A) Predicted 3D structure of eEF1A from T. brucei (TriTrypDB Tb927.10.2100) showing three distinct structural domains (I–III) and the EPG attachment site (Glu362). (B) Proposed pathway for attachment of EPG to eEF1A: PE is attached to Glu362 and subsequently deacylated to EPG.

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The biosynthetic pathway for EPG attachment has not been firmly established. Although an early study proposed that binding of free ethanolamine to eEF1A may represent the first reaction towards a stepwise assembly of EPG [37], the chemical structure of EPG (Fig. 2B) suggests that the entire EPG moiety may derive from phosphatidylethanolamine (PE). Studies using T. brucei parasites defective in PE biosynthesis showed that, indeed, PE is a direct precursor of EPG in T. brucei eEF1A [35]. Based on these findings, we propose a model in which eEF1A is first modified by PE and then becomes deacylated to EPG (Fig. 2B). If correct, such a model would predict that a PElinked eEF1A intermediate might transiently bind to membranes. Surprisingly, although the covalent attachment of EPG to eEF1A was described more than 20 years ago, nothing is known about its biological function. eEF2 and diphthamide The GTPase eEF2 catalyzes the coordinated movement of peptide-tRNA, unloaded tRNA and mRNA, and induces conformational changes in the ribosome (reviewed in [38]). Bacterial EF-G, archaeal aEF2 and eukaryotic eEF2 clearly show structural and functional homologies (Fig. 1). They all consist of six structural domains (I–V and G¢; Fig. 3A) with the binding


pocket for GDP ⁄ GTP being located in domain I [39]. It has been shown that, upon binding of the antifungal inhibitor sordarin, yeast eEF2 can undergo dramatic conformational changes involving rotations of up to 75 of domains IV–V relative to the amino-terminal domains I–II and G¢ through a switch in domain III [40] that may be decisive for its translocation activity. eEF2 was reported to be negatively regulated by phosphorylation by eEF2-kinase leading to a complete arrest of translation elongation (reviewed in [41]). The unique diphthamide [2-(3-carboxyamido-3-(trimethylammonio)propyl)-histidine] modification [42] is conserved from archaea to human but is absent in bacteria (Figs 1 and S4). Diphthamide serves as cellular target for diphtheria toxin from Corynebacterium diphtheriae (reviewed in [43,44]), exotoxin A from Pseudomonas aeruginosa [45,46] and cholix toxin from Vibrio cholerae [47,48]. These toxins catalyze the transfer of ADP-ribose from NAD+ to eEF2-bound diphthamide resulting in irreversible inactivation of eEF2 and cell death. Enzymatic mono-ADP ribosylation is a phylogenetically ancient mechanism to modulate protein function in prokaryotes, eukaryotes and viruses [49–51]. Exotoxin A mimics part of the 80S ribosomal structure and interacts with diphthamide-modified eEF2 leading to its ADP ribosylation [52].

Fig. 3. Attachment of diphthamide to eEF2. (A) 3D structure of eEF2 from S. cerevisiae (PDB, 2P8Z) showing six distinct structural domains (I–V and G¢) and diphthamide attachment to His699. (B) Pathway for diphthamide synthesis: histidine is modified by a reaction sequence involving five separate enzymes (Dph1–5) to diphthine followed by conversion to diphthamide.


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The biosynthesis of diphthamide involves the stepwise addition of different functional groups to the side chain of a distinct histidine residue in eEF2 (His715 in mammals and His699 in S. cerevisiae) by a coordinated action of the conserved enzymes Dph1–Dph5 and a yet unknown amidase (Fig. 3B) [53–58]. The diphthamide modification is located at the tip of domain IV of eEF2 (Fig. 3A) that is supposed to mimic the tRNA anticodon loop [59]. To determine the amino acid requirements of eEF2 for recognition by diphthamide biosynthetic enzymes, site-directed mutagenesis was performed on several residues within the diphthamide-containing loop (Leu693–Gly703) of yeast eEF2. Upon replacement of six residues by alanine, mutated eEF2 proteins were lacking the diphthamide moiety [46]. Similarly, replacement of Gly717 or Gly719 in mammalian eEF2 led to diphtheria toxinresistant cells [60,61]. Despite the fact that this modification was first described more than 30 years ago [42], its role in normal cellular function has remained largely elusive. Systematic mutagenesis of yeast eEF2-His699 showed that the resulting eEF2 proteins were lacking diphthamide and, consequently, were not ADP-ribosylated by diphtheria toxin [62]. Interestingly, the various yeast eEF2 mutants were either lethal indicating a key role of His699 for eEF2 function or led to temperature-sensitive growth of yeast indicating that diphthamide attachment to eEF2 is not strictly required for cell growth [62,63]. The dispensability of diphthamide for eEF2 function was later confirmed by mutagenesis of eEF2-His715 in mammals [64]. Moreover, yeast mutants lacking Dph1, Dph2, Dph4 or Dph5 genes showed no growth phenotypes compared with wild-type cells [58]. The non-essentiality of diphthamide and the Dph enzymes raises the question why such a complex posttranslational modification has been maintained in archaea and eukarya. It has been postulated that essential functions of diphthamide may only become apparent under certain circumstances, e.g. in the context of a multi-cellular organism or during stress conditions [65]. In mouse and human, Dph1 has been identified as a tumor suppressor gene [66–68]. In mice, knockout of one Dph1 allele lead to increased tumor development whereas loss of both Dph1 alleles resulted in death at an early age [69]. Similarly, Dph3 knockout mice showed embryonic lethality [70]. These observations indicate a potential role for diphthamide in the control of tumorigenesis, cell growth and embryonic development. However, the effects caused by loss of dph genes in mammals may be related to other functions of the gene products such as tRNA modification by Dph3 [71]. 2618

As mentioned, the importance of diphthamide in eEF2 function may become apparent during stress conditions [65]. For instance, yeast strains expressing H699N eEF2 or lacking Dph2 or Dph5 are viable but reveal increased frequency in ())1 ribosomal frame shifting [59]. Furthermore, diphthamide has been proposed to protect ribosomes from ribosome-inactivating proteins by showing that cultured Chinese hamster ovary cells lacking the diphthamide biosynthetic enzymes Dph2, Dph3 or Dph5 were threefold more sensitive towards ricin than wild-type cells [65]. After complementation with the corresponding dph genes, the mutant cells gained resistance to ricin. Alternatively, diphthamide may serve as a regulatory modification site of eEF2. It has been previously postulated that ADP ribosylation by diphtheria toxin may represent a normal cellular control mechanism (reviewed in [72]). In mammalian cells, an endogenous ADP-ribosyltransferase activity specific for eEF2 has been described [73–75] that may function in controlling protein synthesis. eIF5A and hypusine For many years, eIF5A was assumed to be involved in translation initiation [76–78]. Only recently, studies in yeast demonstrated that eIF5A promotes translation elongation rather than translation initiation [5,14,79]. eIF5A stimulates translation directly and functions as a general translation elongation factor in a manner determined by its hypusine modification [5]. The unique hypusine [Ne-(4-amino-2-hydroxybutyl)lysine] modification [80] attached to domain I of eIF5A has been found in all eukaryotes examined so far (reviewed in [81,82]) (Fig. 4A). In addition, it also occurs in certain archaea [83] but has not been detected in bacteria. However, in E. coli the conserved lysine residue in domain I of EF-P (Fig. S5) is modified by lysine by a paralog of lysyl-tRNA synthetase. Interestingly, the structure of EF-P mimics that of L-shaped tRNA and its lysylation site (Lys34) corresponds to the tRNA 3¢ end [9]. Domains I and II are highly conserved among all organisms; however, eIF5A and aIF5A lack a carboxyterminal domain III found in bacterial EF-P (see Fig. 1). While the aminoterminal domain I is located close to the aminoacyl acceptor stem of initiator tRNA bound to the P-site of the 70S ribosome, the carboxyterminal domain III of bacterial EF-P is positioned close to the anticodon stem-loop [6]. Hypusine is formed by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOOH) (Fig. 4B).


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Fig. 4. Attachment of hypusine to eIF5A. (A) Predicted 3D structure of human eIF5A (PDB, 1FH4) showing two distinct structural domains (I, II) and the hypusine attachment site (Lys50). (B) Pathway for hypusine synthesis: spermidine is attached to lysine and subsequently modified to hypusine.

Both enzymes are highly conserved among eukaryotes and display similar structural requirements for their substrates, eIF5A-lysine and eIF5A-deoxyhypusine [84–86]. While neither DHS nor DOOH are found in bacteria, a gene homolog for DHS has been identified in archaea. However, it is not clear how hypusinated aIF5A is generated in archaea [87]. Mutations at the hypusine attachment site Lys50 in human eIF5A (Fig. 4A) completely blocked deoxyhypusine synthesis whereas substitutions in its vicinity resulted in reduced efficiency of deoxyhypusine synthesis or inhibition of the hydroxylation reaction catalyzed by DOOH [88]. A truncated peptide consisting of 80 residues of human eIF5A (amino acids 10–90; expressed in E. coli) was nearly as good a substrate as the full-length protein for hypusine attachment [85,86]. Disruption of the eIF5A [89,90] or DHS [91,92] gene results in a lethal phenotype. In contrast, the DOOH gene does not appear to be essential in S. cerevisiae since growth of a DOOH null mutant strain was only slightly reduced compared with the parental strain [93]. However, in multi-cellular organisms such as Caenorhabditis elegans or Drosophila melanogaster inactivation of the DOOH gene was found to be recessively lethal [94,95]. Thus, although in single cell eukaryotes deoxyhypusinated eIF5A is sufficient to perform its

essential cellular functions, multi-cellular eukaryotes require hypusinated eIF5A. In addition to the abovementioned phenotypes, hypusine is necessary for homodimerization of eIF5A and affects its subcellular localization [96,97]. However, the precise mode of eIF5A action and how hypusine modulates eIF5A function remain to be answered. It is possible that eIF5A fulfills the same function as its bacterial ortholog EF-P, which has been shown to catalyze the formation of the first peptide bond in protein synthesis (reviewed in [98]). The recent resolution of its crystal structure [6] has provided new insights into the function of EF-P, indicating that it allows proper positioning of initiator met-tRNA at the P-site of the ribosome in a situation where the E-site of the ribosome is not occupied by unloaded tRNA.

Conclusions We have reviewed the unusual post-translational modifications of three different translation elongation factors that are present in all cells and participate in a conserved mechanistic pathway among eukaryotes and prokaryotes. Though not essential in all organisms (Fig. 1), EPG, diphthamide and hypusine are important to maintain the activity (and probably also the


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proper structure) of acceptor proteins. The biological significance of these modifications may only become evident in vivo or under certain stress or competition conditions, which so far have not been mimicked in the laboratory. In all eukaryotes studied, the function of eIF1A, eEF2 and eIF5A is essential for cell survival. To our knowledge, cross-complementation experiments with paralog prokaryotic and eukaryotic factors have so far not been reported. One possible reason why such experiments may not work would be due to co-evolution of these proteins with their interacting partners which might have given rise to subtle differences that do not allow for cross-complementation of single paralogs in different organisms. Whether EPG, diphthamide and hypusine play a role in protein–protein interactions is unknown. The availability of efficient knockout ⁄ knockin and knockdown techniques using mono- and multi-cellular organisms may allow our knowledge about the importance of these modifications to be extended in the near future. Why are the three modifications EPG, diphthamide and hypusine restricted to single proteins and why are the three modified proteins all involved in elongation of translation? We propose that the modifications are remnants of an evolutionary process that might have been more common in an ancient world, i.e. that multiple proteins were modified by EPG, diphthamide and hypusine. During the course of evolution, however, these modifications may have mostly disappeared, except for the translation elongation proteins e(a)EF1A ⁄ EF-Tu, e(a)EF2 ⁄ EF-G and e(a)IF5A ⁄ EF-P, which are highly conserved between organisms and for which EPG, diphthamide and hypusine may fulfill important functions to enhance accuracy or catalytic activity of enzymes interacting with translating ribosomes. For diphthamide, and in part hypusine, functional essentiality has been demonstrated. In contrast, no functional role has so far been reported for EPG.

Acknowledgements We thank U. Baumann (University of Ko¨ln) and G. Hernandez (McGill University, Montreal) for advice during preparation of the manuscript. E.G. thanks P. Ma¨ser (Swiss Tropical and Public Health Institute, Basel) for support. P.B. thanks G. Moore for stimulation and input and O. Bu¨tikofer for support. Research in our laboratories is supported by Swiss National Science Foundation grants 31003A-130815 to P.B. and 31003A-119996 to M.A. 2620

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Supporting information The following supplementary material is available: Fig. S1. Alignment of primary sequences of eEF1A. Fig. S2. Alignment of partial amino acid sequences of e(a)EF1A ⁄ EF-Tu. Fig. S3. Alignment of partial amino acid sequences of EF-Tu.

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Fig. S4. Alignment of partial amino acid sequences of e(a)EF2 ⁄ EF-G. Fig. S5. Alignment of partial amino acid sequences of e(a)IF5A ⁄ EF-P. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
