Báo cáo khoa học: Unique modifications of translation elongation factors pot

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:
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:
*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
Covalent modifications of proteins often modulate their biological func-
tions or change their subcellular location. Among the many known protein
modifications, three are exceptional in that they only occur on single pro-
teins: ethanolamine phosphoglycerol, diphthamide and hypusine. Remark-
ably, 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.
Introduction
Several hundred protein modifications are known
today, making proteomes far more complex than could
be predicted by the encoding genomes. Covalent modi-
fications modulate the biological functions or change
the subcellular location of proteins and affect interac-
tions 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 multi-
ple amino acid residues [4]. The synthesis and attach-
ment 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 2613
extra means to diversify and develop. While some
modifications are transient and thus depend on rapid
attachment and removal of molecules from target pro-
teins, others are stable and attached to proteins shortly
after their synthesis or before degradation [4].
Among many protein modifications, three are excep-
tional in that they only occur on single proteins: etha-
nolamine 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 eukary-
otes. Single steps of elongation consist of (a) binding of
aminoacyl-tRNAs to the A(minoacyl)-site of the ribo-
some, (b) peptide bond formation with the adjacent
peptide-tRNA at the P(eptidyl)-site and (c) transloca-
tion 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 nowa-
days 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 bio-
logical roles during elongation of translation are as fol-
lows: 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 par-
ticipate in a variety of other functions (so called moon-
lighting 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 elon-
gation, 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 trans-
port aminoacyl-tRNAs to an ancestral membrane-

bound self-folding RNA, which catalyzed peptide bond
formation and constituted the original peptidyltrans-
ferase 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-evo-
lution between proteins and RNA structures, elonga-
tion (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-Phe-
tRNA
Phe
complex [8]. In addition, the crystal structure
of EF-P from Escherichia coli with its post-transla-
tional 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 produc-
tion 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 essen-
tial proteins involved in a highly conserved process,

i.e. elongation of protein translation, and that species-
specific variants of the three proteins have been crys-
tallized 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. Inter-
estingly, 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 differ-
ences: 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 pep-
tide chain elongation in all eukaryotic cells. It interacts
in its GTP-bound form with an aminoacylated tRNA
Unique modifications of translation elongation factors E. Greganova et al.
2614 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS
to mediate binding to the acceptor site of a ribosome
via codon–anticodon interaction. Following ribosome-
dependent 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 reacti-
vate 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 nucleo-
tides 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 elonga-
tion factor 3 [18,19]. Beside its canonical role in pro-
tein synthesis, eEF1A has been shown to also bind to
cytoskeletal proteins and mediate their interactions
[20–22]. This function, which has been localized to
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 bac-
teria. The position of the unique modifications hypusine (Hyp), diphthamide (Dph) and ethanolamine phosphoglycerol (EPG) attached to con-
served 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].
E. Greganova et al. Unique modifications of translation elongation factors
FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2615
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 transduc-
tion 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 con-
served 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. Interest-
ingly, 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 con-
served 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 Haloquad-
ratum 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 eukary-
otes, 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-dimen-
sional 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.
Unique modifications of translation elongation factors E. Greganova et al.
2616 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS
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 PE-
linked 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 move-
ment 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 antifun-
gal inhibitor sordarin, yeast eEF2 can undergo dra-
matic conformational changes involving rotations of
up to 75° of domains IV–V relative to the amino-ter-
minal domains I–II and G¢ through a switch in
domain III [40] that may be decisive for its transloca-
tion activity. eEF2 was reported to be negatively regu-
lated by phosphorylation by eEF2-kinase leading to a
complete arrest of translation elongation (reviewed in
[41]).
The unique diphthamide [2-(3-carboxyamido-3-(trim-
ethylammonio)propyl)-histidine] modification [42] is
conserved from archaea to human but is absent in bac-
teria (Figs 1 and S4). Diphthamide serves as cellular
target for diphtheria toxin from Corynebacterium diph-
theriae (reviewed in [43,44]), exotoxin A from Pseudo-
monas aeruginosa [45,46] and cholix toxin from
Vibrio cholerae [47,48]. These toxins catalyze the trans-
fer of ADP-ribose from NAD
+
to eEF2-bound diph-
thamide resulting in irreversible inactivation of eEF2
and cell death.
Enzymatic mono-ADP ribosylation is a phylogeneti-
cally ancient mechanism to modulate protein function
in prokaryotes, eukaryotes and viruses [49–51]. Exo-
toxin 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.
E. Greganova et al. Unique modifications of translation elongation factors
FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2617
The biosynthesis of diphthamide involves the step-
wise 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 coordi-
nated 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 ala-
nine, mutated eEF2 proteins were lacking the diphtha-
mide moiety [46]. Similarly, replacement of Gly717 or
Gly719 in mammalian eEF2 led to diphtheria toxin-
resistant 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. System-
atic 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 dispens-
ability 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 post-
translational 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 con-
text of a multi-cellular organism or during stress con-
ditions [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 observa-
tions 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 func-
tions of the gene products such as tRNA modification
by Dph3 [71].
As mentioned, the importance of diphthamide in
eEF2 function may become apparent during stress con-
ditions [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 pro-
posed 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 pos-
tulated 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 [N e-(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 modi-
fied 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) corre-
sponds 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 amino-
terminal 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).
Unique modifications of translation elongation factors E. Greganova et al.
2618 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS
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 Caenor-
habditis elegans or Drosophila melanogaster inactiva-
tion 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 above-
mentioned 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 ortho-
log EF-P, which has been shown to catalyze the forma-
tion 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 occu-
pied by unloaded tRNA.
Conclusions
We have reviewed the unusual post-translational modi-
fications of three different translation elongation fac-
tors 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 impor-
tant to maintain the activity (and probably also the
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 modi-
fied to hypusine.
E. Greganova et al. Unique modifications of translation elongation factors
FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2619
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-evolu-
tion of these proteins with their interacting partners
which might have given rise to subtle differences that
do not allow for cross-complementation of single para-
logs in different organisms.

Whether EPG, diphthamide and hypusine play a
role in protein–protein interactions is unknown. The
availability of efficient knockout ⁄ knockin and knock-
down techniques using mono- and multi-cellular
organisms may allow our knowledge about the impor-
tance 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 elonga-
tion of translation? We propose that the modifica-
tions 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 evo-
lution, 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 demon-
strated. 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 stimula-
tion 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.
References
1 Walsh CT, Garneau-Tsodikova S & Gatto GJ Jr (2005)
Protein posttranslational modifications: the chemistry of
proteome diversifications. Angew Chem Int Ed Engl 44,
7342–7372.
2 Seet BT, Dikic I, Zhou MM & Pawson T (2006) Read-
ing protein modifications with interaction domains. Nat
Rev Mol Cell Biol 7, 473–483.
3 Ahrne E, Muller M & Lisacek F (2010) Unrestricted
identification of modified proteins using MS ⁄ MS.
Proteomics 10, 671–686.
4 Yang XJ (2005) Multisite protein modification and
intramolecular signaling. Oncogene 24, 1653–1662.
5 Saini P, Eyler DE, Green R & Dever TE (2009) Hypu-
sine-containing protein eIF5A promotes translation
elongation. Nature 459, 118–121.
6 Blaha G, Stanley RE & Steitz TA (2009) Formation of
the first peptide bond: the structure of EF-P bound to

the 70S ribosome. Science 325, 966–970.
7 Hartman H & Smith TF (2010) GTPases and the origin
of the ribosome. Biol Direct 5, 36.
8 Nakamura Y & Ito K (2003) Making sense of mimic in
translation termination. Trends Biochem Sci 28, 99–105.
9 Yanagisawa T, Sumida T, Ishii R, Takemoto C &
Yokoyama S (2010) A paralog of lysyl-tRNA
synthetase aminoacylates a conserved lysine residue in
translation elongation factor P. Nat Struct Mol Biol 17,
1136–1143.
10 Andersen GR, Nissen P & Nyborg J (2003) Elongation
factors in protein biosynthesis. Trends Biochem Sci 28,
434–441.
11 Le Sourd F, Boulben S, Le Bouffant R, Cormier P,
Morales J, Belle R & Mulner-Lorillon O (2006) eEF1B:
at the dawn of the 21st century. Biochim Biophys Acta
1759, 13–31.
12 Andersen GR, Pedersen L, Valente L, Chatterjee I,
Kinzy TG, Kjeldgaard M & Nyborg J (2000) Structural
basis for nucleotide exchange and competition with
tRNA in the yeast elongation factor complex
eEF1A:eEF1Balpha. Mol Cell 6, 1261–1266.
13 Andersen GR, Valente L, Pedersen L, Kinzy TG &
Nyborg J (2001) Crystal structures of nucleotide
exchange intermediates in the eEF1A-eEF1Balpha
complex. Nat Struct Biol 8, 531–534.
14 Zanelli CF, Maragno AL, Gregio AP, Komili S,
Pandolfi JR, Mestriner CA, Lustri WR & Valentini SR
(2006) eIF5A binds to translational machinery compo-
nents and affects translation in yeast. Biochem Biophys

Res Commun 348, 1358–1366.
15 Carr-Schmid A, Durko N, Cavallius J, Merrick WC &
Kinzy TG (1999) Mutations in a GTP-binding motif of
eukaryotic elongation factor 1A reduce both transla-
tional fidelity and the requirement for nucleotide
exchange. J Biol Chem 274, 30297–30302.
Unique modifications of translation elongation factors E. Greganova et al.
2620 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS
16 Kinzy TG, Freeman JP, Johnson AE & Merrick WC
(1992) A model for the aminoacyl-tRNA binding site of
eukaryotic elongation factor 1 alpha. J Biol Chem 267,
1623–1632.
17 Dinman JD & Kinzy TG (1997) Translational misread-
ing: mutations in translation elongation factor 1alpha
differentially affect programmed ribosomal frameshif-
ting and drug sensitivity. RNA 3, 870–881.
18 Anand M, Chakraburtty K, Marton MJ, Hinnebusch
AG & Kinzy TG (2003) Functional interactions
between yeast translation eukaryotic elongation factor
(eEF) 1A and eEF3. J Biol Chem 278, 6985–6991.
19 Anand M, Balar B, Ulloque R, Gross SR & Kinzy TG
(2006) Domain and nucleotide dependence of the inter-
action between Saccharomyces cerevisiae translation
elongation factors 3 and 1A. J Biol Chem 281,
32318–32326.
20 Liu G, Tang J, Edmonds BT, Murray J, Levin S &
Condeelis J (1996) F-actin sequesters elongation factor
1alpha from interaction with aminoacyl-tRNA in a
pH-dependent reaction. J Cell Biol 135, 953–963.
21 Gross SR & Kinzy TG (2005) Translation elongation

factor 1A is essential for regulation of the actin cyto-
skeleton and cell morphology. Nat Struct Mol Biol 12,
772–778.
22 Gross SR & Kinzy TG (2007) Improper organization of
the actin cytoskeleton affects protein synthesis at initia-
tion. Mol Cell Biol 27, 1974–1989.
23 Panasyuk G, Nemazanyy I, Filonenko V, Negrutskii B
& El’skaya AV (2008) A2 isoform of mammalian trans-
lation factor eEF1A displays increased tyrosine phos-
phorylation and ability to interact with different
signalling molecules. Int J Biochem Cell Biol 40, 63–71.
24 Khacho M, Mekhail K, Pilon-Larose K, Pause A, Cote
J & Lee S (2008) eEF1A is a novel component of the
mammalian nuclear protein export machinery. Mol Biol
Cell 19, 5296–5308.
25 Bouzaidi-Tiali N, Aeby E, Charriere F, Pusnik M &
Schneider A (2007) Elongation factor 1a mediates the
specificity of mitochondrial tRNA import in T. brucei.
EMBO J 26, 4302–4312.
26 Peters HI, Chang YW & Traugh JA (1995) Phosphory-
lation of elongation factor 1 (EF-1) by protein kinase C
stimulates GDP ⁄ GTP-exchange activity. Eur J Biochem
234, 550–556.
27 Chang YW & Traugh JA (1998) Insulin stimulation of
phosphorylation of elongation factor 1 (eEF-1) enhances
elongation activity. Eur J Biochem 251, 201–207.
28 Cavallius J, Zoll W, Chakraburtty K & Merrick WC
(1993) Characterization of yeast EF-1alpha: non-conser-
vation of post-translational modifications. Biochim
Biophys Acta 1163, 75–80.

29 Sherman M & Sypherd PS (1989) Role of lysine methyl-
ation in the activities of elongation factor 1 alpha. Arch
Biochem Biophys 275, 371–378.
30 Zobel-Thropp P, Yang MC, Machado L & Clarke S
(2000) A novel post-translational modification of
yeast elongation factor 1A. J Biol Chem 275,
37150–37158.
31 Ejiri S (2002) Moonlighting functions of polypeptide
elongation factor 1: from actin bundling to zinc finger
protein R1-associated nuclear localization. Biosci
Biotechnol Biochem 66, 1–21.
32 Whiteheart SW, Shenbagamurthi P, Chen L, Cotter RJ
& Hart GW (1989) Murine elongation factor 1alpha is
posttranslationally modified by novel amide-linked etha-
nolamine-phosphoglycerol moieties. J Biol Chem 264,
14334–14341.
33 Rosenberry TL, Krall JA, Dever TE, Haas R,
Louvard D & Merrick WC (1989) Biosynthetic
incorporation of [3H]ethanolamine into protein synthe-
sis elongation factor 1alpha reveals a new post-transla-
tional protein modification. J Biol Chem 264, 7096–
7099.
34 Ransom WD, Lao P-C, Gage DA & Boss WF (1998)
Phosphoglycerylethanolamine posttranslational modifi-
cation of plant eukaryotic elongation factor 1alpha.
Plant Physiol 117, 949–960.
35 Signorell A, Jelk J, Rauch M & Bu
¨
tikofer P (2008)
Phosphatidylethanolamine is the precursor of the

ethanolamine phosphoglycerol moiety bound to eukary-
otic elongation factor 1A. J Biol Chem 283, 20320–
20329.
36 Greganova E, Heller M & Bu
¨
tikofer P (2010) A struc-
tural domain mediates attachment of ethanolamine
phosphoglycerol to eukaryotic elongation factor 1A in
Trypanosoma brucei. PLoS ONE 5, e9486.
37 Whiteheart SW & Hart GW (1994) Incorporation of
[3H]ethanolamine into a single cytosolic protein in a cell
free system: ethanolaminylation of EF-1 alpha in vitro.
Arch Biochem Biophys 309, 387–391.
38 Jorgensen R, Merrill AR & Andersen GR (2006) The
life and death of translation elongation factor 2.
Biochem Soc Trans 34, 1–6.
39 Yates SP, Jorgensen R, Andersen GR & Merrill AR
(2006) Stealth and mimicry by deadly bacterial toxins.
Trends Biochem Sci 31, 123–133.
40 Jorgensen R, Ortiz PA, Carr-Schmid A, Nissen P,
Kinzy TG & Andersen GR (2003) Two crystal struc-
tures demonstrate large conformational changes in the
eukaryotic ribosomal translocase. Nat Struct Biol 10,
379–385.
41 Browne GJ & Proud CG (2002) Regulation of peptide-
chain elongation in mammalian cells. Eur J Biochem
269, 5360–5368.
42 Van Ness BG, Howard JB & Bodley JW (1980)
ADP-ribosylation of elongation factor 2 by diphtheria
toxin. Isolation and properties of the novel ribosyl-

amino acid and its hydrolysis products. J Biol Chem
255, 10717–10720.
E. Greganova et al. Unique modifications of translation elongation factors
FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2621
43 Pappenheimer AM Jr (1977) Diphtheria toxin. Annu
Rev Biochem 46, 69–94.
44 Oppenheimer NJ & Bodley JW (1981) Diphtheria toxin.
Site and configuration of ADP-ribosylation of diphtha-
mide in elongation factor 2. J Biol Chem 256,
8579–8581.
45 Iglewski BH & Kabat D (1975) NAD-dependent inhibi-
tion of protein synthesis by Pseudomonas aeruginosa
toxin. Proc Natl Acad Sci USA 72, 2284–2288.
46 Zhang Y, Liu S, Lajoie G & Merrill AR (2008) The
role of the diphthamide-containing loop within eukary-
otic elongation factor 2 in ADP-ribosylation by Pseudo-
monas aeruginosa exotoxin A. Biochem J 413, 163–174.
47 Liu S & Leppla SH (2003) Retroviral insertional muta-
genesis identifies a small protein required for synthesis
of diphthamide, the target of bacterial ADP-ribosylat-
ing toxins. Mol Cell 12, 603–613.
48 Jorgensen R, Purdy AE, Fieldhouse RJ, Kimber MS,
Bartlett DH & Merrill AR (2008) Cholix toxin, a novel
ADP-ribosylating factor from Vibrio cholerae. J Biol
Chem 283, 10671–10678.
49 Pallen MJ, Lam AC, Loman NJ & McBride A (2001)
An abundance of bacterial ADP-ribosyltransferases –
implications for the origin of exotoxins and their human
homologues. Trends Microbiol 9, 302–307; discussion
308.

50 Krueger KM & Barbieri JT (1995) The family of bacte-
rial ADP-ribosylating exotoxins. Clin Microbiol Rev 8,
34–47.
51 Corda D & Di Girolamo M (2003) Functional aspects
of protein mono-ADP-ribosylation. EMBO J 22,
1953–1958.
52 Jorgensen R, Merrill AR, Yates SP, Marquez VE,
Schwan AL, Boesen T & Andersen GR (2005) Exotoxin
A-eEF2 complex structure indicates ADP ribosylation
by ribosome mimicry. Nature 436, 979–984.
53 Moehring JM, Moehring TJ & Danley DE (1980) Post-
translational modification of elongation factor 2 in
diphtheria-toxin-resistant mutants of CHO-K1 cells.
Proc Natl Acad Sci USA 77, 1010–1014.
54 Dunlop PC & Bodley JW (1983) Biosynthetic labeling
of diphthamide in Saccharomyces cerevisiae. J Biol
Chem 258, 4754–4758.
55 Moehring TJ, Danley DE & Moehring JM (1984)
In vitro biosynthesis of diphthamide, studied with
mutant Chinese hamster ovary cells resistant to
diphtheria toxin. Mol Cell Biol 4, 642–650.
56 Chen JY, Bodley JW & Livingston DM (1985) Diphthe-
ria toxin-resistant mutants of Saccharomyces cerevisiae.
Mol Cell Biol 5, 3357–3360.
57 Chen JY & Bodley JW (1988) Biosynthesis of diphtha-
mide in Saccharomyces cerevisiae. Partial purification
and characterization of a specific S-adenosylmethionine:
elongation factor 2 methyltransferase. J Biol Chem 263,
11692–11696.
58 Liu S, Milne GT, Kuremsky JG, Fink GR & Leppla

SH (2004) Identification of the proteins required for
biosynthesis of diphthamide, the target of bacterial
ADP-ribosylating toxins on translation elongation fac-
tor 2. Mol Cell Biol 24, 9487–9497.
59 Ortiz PA, Ulloque R, Kihara GK, Zheng H & Kinzy
TG (2006) Translation elongation factor 2 anticodon
mimicry domain mutants affect fidelity and diphtheria
toxin resistance. J Biol Chem 281 , 32639–32648.
60 Foley BT, Moehring JM & Moehring TJ (1995)
Mutations in the elongation factor 2 gene which confer
resistance to diphtheria toxin and Pseudomonas
exotoxin A. Genetic and biochemical analyses. J Biol
Chem 270, 23218–23225.
61 Kohno K & Uchida T (1987) Highly frequent single
amino acid substitution in mammalian elongation factor
2 (EF-2) results in expression of resistance to EF-2-ADP-
ribosylating toxins. J Biol Chem 262, 12298–12305.
62 Kimata Y & Kohno K (1994) Elongation factor 2
mutants deficient in diphthamide formation show
temperature-sensitive cell growth. J Biol Chem 269,
13497–13501.
63 Phan LD, Perentesis JP & Bodley JW (1993) Saccharo-
myces cerevisiae elongation factor 2. Mutagenesis of the
histidine precursor of diphthamide yields a functional
protein that is resistant to diphtheria toxin. J Biol Chem
268, 8665–8668.
64 Ivankovic M, Rubelj I, Matulic M, Reich E & Brdar B
(2006) Site-specific mutagenesis of the histidine precur-
sor of diphthamide in the human elongation factor-2
gene confers resistance to diphtheria toxin. Mutat Res

609, 34–42.
65 Gupta PK, Liu S, Batavia MP & Leppla SH (2008) The
diphthamide modification on elongation factor-2
renders mammalian cells resistant to ricin. Cell Micro-
biol 10, 1687–1694.
66 Phillips NJ, Zeigler MR & Deaven LL (1996) A cDNA
from the ovarian cancer critical region of deletion on
chromosome 17p13.3. Cancer Lett 102, 85–90.
67 Bruening W, Prowse AH, Schultz DC, Holgado-Madru-
ga M, Wong A & Godwin AK (1999) Expression of
OVCA1, a candidate tumor suppressor, is reduced in
tumors and inhibits growth of ovarian cancer cells.
Cancer Res 59, 4973–4983.
68 Schultz DC, Vanderveer L, Berman DB, Hamilton TC,
Wong AJ & Godwin AK (1996) Identification of two
candidate tumor suppressor genes on chromosome
17p13.3. Cancer Res 56, 1997–2002.
69 Chen CM & Behringer RR (2004) Ovca1 regulates cell
proliferation, embryonic development, and tumorigene-
sis. Genes Dev 18, 320–332.
70 Liu S, Wiggins JF, Sreenath T, Kulkarni AB, Ward JM
& Leppla SH (2006) Dph3, a small protein required for
diphthamide biosynthesis, is essential in mouse develop-
ment. Mol Cell Biol 26, 3835–3841.
Unique modifications of translation elongation factors E. Greganova et al.
2622 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS
71 Huang B, Johansson MJ & Bystrom AS (2005) An
early step in wobble uridine tRNA modification
requires the Elongator complex. RNA 11, 424–436.
72 Collier RJ (1975) Diphtheria toxin: mode of action and

structure. Bacteriol Rev 39, 54–85.
73 Lee H & Iglewski WJ (1984) Cellular ADP-ribosyltrans-
ferase with the same mechanism of action as diphtheria
toxin and Pseudomonas toxin A. Proc Natl Acad Sci
USA 81, 2703–2707.
74 Sitikov AS, Davydova EK, Bezlepkina TA, Ovchinni-
kov LP & Spirin AS (1984) Eukaryotic elongation fac-
tor 2 loses its non-specific affinity for RNA and leaves
polyribosomes as a result of ADP-ribosylation. FEBS
Lett 176, 406–410.
75 Sayhan O, Ozdemirli M, Nurten R & Bermek E (1986)
On the nature of cellular ADP-ribosyltransferase from
rat liver specific for elongation factor 2. Biochem
Biophys Res Commun 139, 1210–1214.
76 Benne R, Brown-Luedi ML & Hershey JW (1978)
Purification and characterization of protein synthesis
initiation factors eIF-1, eIF-4C, eIF-4D, and eIF-5 from
rabbit reticulocytes. J Biol Chem 253, 3070–3077.
77 Smit-McBride Z, Schnier J, Kaufman RJ & Hershey
JW (1989) Protein synthesis initiation factor eIF-4D.
Functional comparison of native and unhypusinated
forms of the protein. J Biol Chem 264, 18527–18530.
78 Park MH (1989) The essential role of hypusine in
eukaryotic translation initiation factor 4D (eIF-4D).
Purification of eIF-4D and its precursors and compari-
son of their activities. J Biol Chem 264, 18531–18535.
79 Gregio AP, Cano VP, Avaca JS, Valentini SR & Zanelli
CF (2009) eIF5A has a function in the elongation step
of translation in yeast. Biochem Biophys Res Commun
380, 785–790.

80 Shiba T, Mizote H, Kaneko T, Nakajima T & Kakim-
oto Y (1971) Hypusine, a new amino acid occurring in
bovine brain. Isolation and structural determination.
Biochim Biophys Acta 244, 523–531.
81 Park MH, Lee YB & Joe YA (1997) Hypusine is essen-
tial for eukaryotic cell proliferation. Biol Signals 6,
115–123.
82 Chen KY & Liu AY (1997) Biochemistry and function
of hypusine formation on eukaryotic initiation factor
5A. Biol Signals 6, 105–109.
83 Bartig D, Lemkemeier K, Frank J, Lottspeich F &
Klink F (1992) The archaebacterial hypusine-containing
protein. Structural features suggest common ancestry
with eukaryotic translation initiation factor 5A. Eur J
Biochem 204, 751–758.
84 Wolff EC, Kang KR, Kim YS & Park MH (2007)
Posttranslational synthesis of hypusine: evolutionary
progression and specificity of the hypusine modification.
Amino Acids 33, 341–350.
85 Joe YA & Park MH (1994) Structural features of the
eIF-5A precursor required for posttranslational
synthesis of deoxyhypusine. J Biol Chem 269,
25916–25921.
86 Kang KR, Kim YS, Wolff EC & Park MH (2007)
Specificity of the deoxyhypusine hydroxylase-eukaryotic
translation initiation factor (eIF5A) interaction: identifi-
cation of amino acid residues of the enzyme required
for binding of its substrate, deoxyhypusine-containing
eIF5A. J Biol Chem 282, 8300–8308.
87 Park MH, Nishimura K, Zanelli CF & Valentini SR

(2010) Functional significance of eIF5A and its
hypusine modification in eukaryotes. Amino Acids 38,
491–500.
88 Cano VS, Jeon GA, Johansson HE, Henderson CA,
Park JH, Valentini SR, Hershey JW & Park MH (2008)
Mutational analyses of human eIF5A-1 – identification
of amino acid residues critical for eIF5A activity and
hypusine modification. FEBS J
275, 44–58.
89 Schnier J, Schwelberger HG, Smit-McBride Z, Kang
HA & Hershey JW (1991) Translation initiation factor
5A and its hypusine modification are essential for cell
viability in the yeast Saccharomyces cerevisiae. Mol Cell
Biol 11, 3105–3114.
90 Wohl T, Klier H, Ammer H, Lottspeich F & Magdolen
V (1993) The HYP2 gene of Saccharomyces cerevisiae is
essential for aerobic growth: characterization of differ-
ent isoforms of the hypusine-containing protein Hyp2p
and analysis of gene disruption mutants. Mol Gen Genet
241, 305–311.
91 Sasaki K, Abid MR & Miyazaki M (1996) Deoxyhypu-
sine synthase gene is essential for cell viability in the yeast
Saccharomyces cerevisiae. FEBS Lett 384, 151–154.
92 Park MH, Joe YA & Kang KR (1998) Deoxyhypusine
synthase activity is essential for cell viability in the yeast
Saccharomyces cerevisiae. J Biol Chem 273 , 1677–1683.
93 Park JH, Aravind L, Wolff EC, Kaevel J, Kim YS &
Park MH (2006) Molecular cloning, expression, and
structural prediction of deoxyhypusine hydroxylase: a
HEAT-repeat-containing metalloenzyme. Proc Natl

Acad Sci USA 103, 51–56.
94 Sugimoto A (2004) High-throughput RNAi in Caenor-
habditis elegans: genome-wide screens and functional
genomics. Differentiation 72, 81–91.
95 Spradling AC, Stern D, Beaton A, Rhem EJ, Laverty
T, Mozden N, Misra S & Rubin GM (1999) The
Berkeley Drosophila Genome Project gene disruption
project: single P-element insertions mutating 25% of
vital Drosophila genes. Genetics 153, 135–177.
96 Lee SB, Park JH, Kaevel J, Sramkova M, Weigert R
& Park MH (2009) The effect of hypusine modification
on the intracellular localization of eIF5A. Biochem
Biophys Res Commun 383, 497–502.
97 Gentz PM, Blatch GL & Dorrington RA (2009)
Dimerization of the yeast eukaryotic translation
initiation factor 5A requires hypusine and is RNA
dependent. FEBS J 276, 695–706.
E. Greganova et al. Unique modifications of translation elongation factors
FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2623
98 Liljas A (2009) Leaps in translational elongation.
Science 326, 677–678.
99 DeLano WL (2008) The PyMOL Molecular Graphics
System. DeLano Scientific LLC, Palo Alto, CA, USA;
.
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.
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.
Unique modifications of translation elongation factors E. Greganova et al.
2624 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS