Novel complexes of mammalian translation elongation factor
eEF1AÆGDP with uncharged tRNA and aminoacyl-tRNA synthetase
Implications for tRNA channeling
Zoya M. Petrushenko, Tatyana V. Budkevich, Vyacheslav F. Shalak, Boris S. Negrutskii
and Anna V. El’skaya
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev, Ukraine
Multimolecular complexes involving the eukaryotic
elongation factor 1A (eEF1A) have been suggested to play
an important role in the channeling (vectorial transfer) of
tRNA during protein synthesis [Negrutskii, B.S. & El’skaya,
A.V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47–78].
Recently we have demonstrated that besides performing its
canonical function of forming a ternary complex with GTP
and aminoacyl-tRNA, the mammalian eEF1A can produce
a noncanonical ternary complex with GDP and uncharged
tRNA [Petrushenko, Z.M., Negrutskii, B.S., Ladokhin,
A.S., Budkevich, T.V., Shalak, V.F. & El’skaya, A.V. (1997)
FEBS Lett. 407, 13–17]. The [eEF1AÆGDPÆtRNA] complex
has been hypothesized to interact with aminoacyl-tRNA
synthetase (ARS) resulting in a quaternary complex where
uncharged tRNA is transferred to the enzyme for amino-
acylation. Here we present the data on association of the
[eEF1AÆGDPÆtRNA] complex with phenylalanyl-tRNA
synthetase (PheRS), e.g. the formation of the above
quaternary complex detected by the gel-retardation and
surface plasmon resonance techniques. To estimate the
stability of the novel ternary and quaternary complexes of
eEF1A the fluorescence method and BIAcore analysis were
used. The dissociation constants for the [eEF1AÆGDPÆ
tRNA] and [eEF1AÆGDPÆtRNA
Phe

which a compartmentalization of the translation apparatus
is of particular importance. There is an increasing body of
evidence for special structural organization of the protein
synthesis machinery in the higher eukaryotic cells. The
existence of multimolecular complexes of ARS [1], initiation
factors [2] and eEF1 [3,4], ribosome–ARS interactions [5–7],
and the association of translation components with cyto-
skeletal framework [8] are among the important signs of the
protein synthesis compartmentalization. Moreover, detailed
fluorescence-based measurements of translation in living
dendrites have visualized the mammalian protein synthesis
compartments in situ [9].
An important mechanism to put into effect the potential
advantages of the compartmentalization is thought to be a
channeling (vectorial transfer) of aminoacyl-tRNA/tRNA
from ARS to the elongation factor, ribosome and back to
ARS without dissociation into the surrounding medium
[10,11]. The channeling influences positively the transla-
tional efficiency because the number of nonspecific
searches is diminished, the effective concentrations of
translational components are increased and the leakage of
important compounds to another metabolic processes is
hampered [12]. The channeling is a mechanism operating
by the formation of intermediate complexes between
subsequent participants of the metabolic pathway. Deut-
scher and coauthors revealed that aminoacyl-tRNA and
tRNA were never free in the cytoplasm of the eukaryotic
cell [10–12]. ARS and eEF1A are supposed to play a main
role in the tRNA sequestering during the mammalian
translation [13].

(3) fi [eEF1AÆGTPÆaminoacyl-tRNA] (4) fi [ribosomal
AsiteÆaminoacyl-tRNA] (5) fi [ribosomal P siteÆ
peptidyl-tRNA] (6) fi [ribosomal E siteÆtRNA] (1).
The existence of complexes 1, 4, 5 and 6 was well
documented and considered in all textbook schemes of
protein synthesis. The formation of noncanonical complex 2
has been demonstrated recently [16] but its thermodynamic
stability has not been determined. The idea of noncanonical
quaternary complex 3 assembling was based on the
stimulatory effect of eEF1AÆGDP on the activity of several
ARS [14], however, it remains to be shown directly.
In this work, the formation of a specific complex of
[eEF1AÆGDPÆtRNA] with PheRS was shown by the gel-
shift assay and surface plasmon resonance technique. High
stability of both novel ternary and quaternary complexes
of eEF1AÆGDP, [eEF1AÆGDPÆtRNA] and [eEF1AÆGDPÆ
tRNA
Phe
ÆPheRS], was observed, the dissociation constants
being determined as 20 n
M
and 9 n
M
, respectively. The
BIAcore analysis revealed a direct protein–protein interac-
tion within the quaternary complex 3. The sequence of
events in the channeled elongation cycle of protein synthesis
is discussed considering a putative supercomplex of ARS
and GDP/GTP exchanging subunits of eEF1.
MATERIALS AND METHODS

using the combination of gel-filtration and ion-exchange
chromatography as previously described [19]. GDP/
[
3
H]GDP exchange on the eEF1A molecule was performed
as described [19]. The purity of the enzymes was more than
95% according to the SDS/PAGE.
Preparation of bacterial EF1AÆGTP
To obtain the GTP form of bacterial EF1A, the factor was
incubated with 100 l
M
GTP in the incubation mixture
containing 25 m
M
Tris/HCl, pH 7.5, 50 m
M
NH
4
Cl, 10 m
M
MgCl
2
,1m
M
dithiothreitol, 0.5 m
M
EDTA in the presence
of 30 lgÆmL
)1
phosphoenolpyruvatekinaseand2m

3
,pH 8.1,2m
M
MgCl
2
,25m
M
KCl, 20%
glycerol, 10 l
M
phenylmethanesulfonyl fluoride and 2 m
M
dithiothreitol at 4 °C. The stock solution of FITC was
added to the final concentration of 0.05 mgÆmL
)1
and the
incubation was continued for 40 min at 28 °C. The reaction
was quenched by addition of 2
M
NH
4
Cl (final concentra-
tion 50 m
M
) and the protein was separated from the dye by
gel-filtration on Sephadex G-25.
To obtain eEF1AÆGMP-PNP, the factor was incubated
with 200 l
M
GMP-PNP in the incubation mixture contain-

200 l
M
GDP (GMP-PNP) and 0.2 l
M
FITC-eEF1AÆGDP
(FITC-eEF1AÆGMP-PNP) at +24 °C. FITC-eEF1AÆGDP
or FITC-eEF1AÆGMP-PNP were titrated by increasing
concentrations of tRNA to measure K
d
of the [eEF1AÆGDP/
GMP-PNPÆtRNA] complex. An increase in the mixture
volume after tRNA addition did not exceed 3–5%. The data
were corrected for the background fluorescence and dilution.
To confirm complex formation, the polarization value
was determined after each tRNA addition. When plane
4812 Z. M. Petrushenko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
polarized light is used to excite a fluorophore, molecules in
which the absorption oscillators are orientated parallel to
the direction of polarization will excite preferentially. The
polarized components of the emission can be used to
calculate a polarization value P ¼ I
||
– I
^
/I
||
+ I
^
(where
I

À 1 ð1Þ
where I
^
norm is the normalized intensity, I
0
?
is the fluor-
escence intensity before tRNA addition, I
tRNA
?
is the
intensity at given tRNA concentration. Data were curve-
fitted by nonlinear least squares to a bimolecular binding
isotherm according to the expression:
I
?
norm ¼ I
fin
?
 C=K
d
þ C ð2Þ
where I
fin
?
is the normalized intensity at final point of the
titration curve, C is the tRNA concentration, K
d
is the
dissociation constant.

M
EDTA and 1 m
M
dithiothreitol. Protein bands were stained with Coomassie
brilliant blue.
The formation of the complex of [
32
P]tRNA
Phe
with
eEF1A and/or PheRS was studied on 0.7% agarose gel.
Three picomoles of tRNA were incubated with 10 pmol of
protein (eEF1A, PheRS or their mixture) at 37 °Cfor
10 min in 15 lLof25m
M
Hepes/KOH, pH 7.6, 5 m
M
MgCl
2
,100m
M
KCl, 10% glycerol, 2 m
M
dithiothreitol
and 100 l
M
GDP. The electrophoresis was run at 20 VÆcm
)1
(50 m
M

)1
). Unreacted
N-hydroxysuccinimide ester groups were quenched by a
30-lL injection of 1
M
ethanolamine/HCl, pH 8.0. The
final level of PheRS immobilization was about 2500
resonance units (RU). Bovine catalase (2500 RU) was
immobilized to the sensor chip in the same way. While
studying the binding kinetics by BIAcore technique there
is a danger of deviations from the real data in case of
high surface density of an immobilized ligand. The mass
transport effect was hypothesized to reduce the effective
binding affinity for a soluble analyte [23]. However, a
comparative analysis [24] of the binding data for
immobilized influenza virus N9 neuraminidase (3000
RU surface density) with molecular mass 190 000 Da
(close to PheRS) and the Fab fragment of monoclonal
antibody of 50 000 Da (equal to eEF1A) with and
without the mass transport correction term at a flow
rate of 50 lLÆmin
)1
showed that there was no significant
difference in the fits indicating, in turn, that the values
measured at such a high flow rate did not contain
significant contribution from the mass transport.
To produce so-called ÔblankÕ chip for the assessment of
nonspecific adsorption of the analyte onto the sensing
surface the sensor chip was activated as described above
with the subsequent quenching of the active groups of

in the same buffer flow for 10 min. KCl (0.5
M
)wasusedto
regenerate a sensor chip after each binding event. The
concentration of the ternary complex was set by eEF1A
concentration.
BIAcore evaluation
The kinetic parameters were calculated using the kinetics
evaluation software package
BIAEVALUATION
3.0 (Pharma-
cia Biosensor). The theory of BIAcore measurement
technique and calculations has been extensively described
[25]. The formation of a surface-bound quaternary com-
plex [eEF1AÆGDPÆtRNAÆPheRS] was treated using
Eqn (3):
A+B À!
k
a
AB À!
k
d
A+B ð3Þ
where A corresponds to the immobilized ligand (PheRS),
B corresponds to analyte (eEF1AÆGDP or [eEF1AÆGDPÆ
tRNA]), k
a
is the association rate constant (
M
)1

5% PAGE (Fig. 1). Under the conditions described in detail
in Materials and methods, eEF1AÆGDP moves rather
slowly (Fig. 1, lane 1) due to its high positive charge. It
did not fully enter the gel even after 6 h of electrophoresis.
As expected, the binding of negatively charged tRNA
during complex formation accelerates the protein band
movement (lanes 2–5). Lane 2 also shows that only at the
ratiooffactortotRNAlessthan2:1apartof
eEF1AÆGDP remains on the start. Thus, practically all
molecules of eEF1AÆGDP were found in the complex and
the amount of inactive eEF1A molecules being negligible.
The [eEF1AÆGDPÆtRNA] complex was shown earlier by
several independent qualitative methods [16]. Here its
formation during the factor titration with tRNA was
confirmed by the fluorescence polarization technique
(Fig. 2A). Indeed, gradual increase in the fluorescence
polarization seen upon the addition of tRNA shows a
change in the rotational mobility of the FITC-eEF1AÆGDP
in the free and tRNA-complexed state. The perpendicular
component of fluorescence intensity (I
^
) was normalized as
described in Materials and methods. To determine K
d
of the
[eEF1AÆGDPÆtRNA] complex the experimental points were
fit to a bimolecular binding isotherm (Fig. 2B) according to
Eqn (2). K
d
for this complex was estimated to be

intensity (B) of 0.2 l
M
FITC-eEF1AÆGDP were recorded in the pres-
ence of indicated tRNA concentrations (0–0.5 l
M
final) as described in
Materials and methods. Reactions were allowed to reach equilibrium
and data were corrected for the background fluorescence and probe
dilution.
Fig. 1. Electrophoresis of eEF1AÆGDP in nondenaturing conditions in
the presence of different tRNA concentrations. eEF1A (10 l
M
)and
indicated amounts of tRNA were incubated 10 min as described in
Materials and methods and the mixture was applied to 5% poly-
acrylamide gel. Electrophoresis was performed for 6 h at 4 °C(40 mA,
100 V) in a buffer containing 100 m
M
Bes, pH 6.8, 10% glycerol,
10 l
M
GDP, 0.5 m
M
EDTA and 1 m
M
dithiothreitol. Protein bands
were visualized by staining with Coomassie brilliant blue.
4814 Z. M. Petrushenko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
[eEF1AÆGDPÆtRNAÆPheRS] complex formation was ana-
lyzed by the gel-retardation assay in 0.7% agarose (Fig. 3).

complex
The stability of the [eEF1AÆGDPÆtRNA
Phe
ÆPheRS] com-
plex was evaluated by the surface plasmon resonance
technique. The BIAcore instrument detects changes in the
surface plasmon resonance to monitor the interaction of
an immobilized ligand with analyte molecules in flow
solution [31]. PheRS was the immobilized ligand in all
experiments because the immobilization of eEF1A led to
a significant loss of its ability to bind tRNA. Therefore,
the ternary [eEF1AÆGDPÆtRNA
Phe
] complex was pre-
formed for 4 min at 25 °C in the running buffer and
injected as analyte. To estimate the contribution of
nonspecific adsorption property of the sensor surface,
control injections of the ternary complex over a blank
chip (see Materials and methods) were performed. A
background signal was automatically subtracted from the
sensograms obtained with immobilized PheRS. The spe-
cificity of the ligand–analyte interaction was verified by
the immobilization of bovine catalase instead of PheRS
over the sensor chip with subsequent injection of
eEF1AÆGDP in flow buffer. It resulted in a signal equal
to the control injection over a blank chip under the same
experimental conditions (data not shown).
Figure 5 shows the increase in the chip response level
upon addition of various concentrations of the [eEF1AÆ
GDPÆtRNA

package. The apparent K
d
for the [eEF1AÆGDPÆPheRS]
complex formation was 21 n
M
. The high affinity of eEF1A
for PheRS may be the reason of their co-purification from
rabbit liver extract during several chromatographic steps
Fig. 3. Nondenaturing agarose electrophoresis assay of the
[
32
P]tRNA
Phe
binding to PheRS and eEF1AÆGDP. tRNA
Phe
(3 pmol)
was incubated with 10 pmol of PheRS (lanes 1, 4) or the mixture of
10 pmol of PheRS and 10 pmol of eEF1A (lanes 2, 5) at 37 °Cfor
10 min. The electrophoresis was run for 2 h at +4 °C in 0.7% agarose
gel. Lane 3 shows [
32
P]tRNA
Phe
alone. The proteins were stained by
Coomassieblue(lanes1,2).[
32
P]tRNA
Phe
was visualized by autora-
diography (lanes 3, 4, 5). To save space, the tRNA

us to propose the tRNA channeling scheme in detail
(Fig. 7).
Taking into account rather low affinity of tRNA for the
E site of 80S ribosomes (the apparent K
d
is about 600 n
M
[33]), it is plausible to assume that the transfer of tRNA
from the E site to eEF1AÆGDP occurs due to the affinity
gradient (K
d
for [eEF1AÆGDPÆtRNA] is 20 n
M
, this study).
Furthermore, the ARS affinity for [eEF1AÆGDPÆtRNA] (K
d
is 9 n
M
, this study) is higher than that for free tRNA (K
d
in
the range of 100–200 n
M
[34,35]), which makes association
of the enzyme with tRNA bound to eEF1AÆGDP thermo-
dynamically favorable. In this quaternary complex, a
transfer of tRNA from the factor to ARS may occur. As
the quaternary complex [eEF1AÆGDPÆtRNAÆARS] (B) is
stabilized by the protein–protein and protein–tRNA inter-
actions, eEF1AÆGDP, being in the quaternary complex,

)3
9 · 10
)9
[eEF1AÆGDPÆPheRS] 3.8 · 10
5
0.8 · 10
)3
21 · 10
)9
Fig. 7. Scheme showing the tRNA/aminoacyl-tRNA channeling in the
translation elongation cycle. d, amino acid; small and large triangles,
tRNA and eEF1Ba, respectively.
Fig. 5. Biosensor assay of the quaternary [eEF1AÆGDPÆtRNA
Phe
Æ
PheRS] complex formation. PheRS was immobilized on the chip
as described in Materials and methods. Injections of the
[eEF1AÆGDPÆtRNA
Phe
] complex at concentrations of 60, 80, 125, 150,
250 and 500 n
M
(curves from bottom to top) were carried out for 200 s
at flow rate of 50 lLÆmin
)1
with the following dissociation of the
quaternary complex for 10 min. The sensograms show the kinetics of
the [eEF1AÆGDPÆtRNA
Phe
] complex binding to immobilized PheRS

tRNAÆARS] dissociates rapidly giving the canonical ternary
complex [eEF1AÆGTPÆaminoacyl-tRNA] (F) and free ARS.
The scheme proposed and the results reported in this
paper are in good agreement with the observation that
tRNA in the eukaryotic cell is always bound to some
protein [11], never being in a ÔfreeÕ state. Further verification
of the sequence of events during tRNA/aminoacyl-tRNA
channeling involving the ARS molecule, as well as the
elucidation of eEF1AÆGDP action during dissociation of
deacylated tRNA from the E site of 80S ribosome is
presently underway.
ACKNOWLEDGMENT
We thank Ivan Gout (the Ludwig Institute for Cancer Research,
London, UK) for permanent support in BIAcore experiments and
Marc Mirande (Laboratoire d’Enzymologie et Biochimie Structu-
rales, CNRS, Gif-sur-Yvette, France) for helpful comments on the
manuscript. This work was supported by International Association
for the Promotion of Cooperation with Scientists from the New
Independent States of the Former Soviet Union (INTAS) Grant
96–1594 and by Ministry for Science and Technologies of Ukraine
Grants 5.4/73 and 5.7/0003. Z.M.P. was supported in part by the
Wellcome Trust Research Travel Grant and FEBS Short-term
Fellowship.
REFERENCES
1. Mirande, M. (1991) Aminoacyl-tRNA synthetase family from
prokaryotes and eukaryotes: structural domains and their impli-
cations. Prog. Nucleic Acids Res. Mol. Biol. 40, 95–142.
2. Asano, K., Clayton, J., Shalev, A. & Hinnebusch, A.G. (2000) A
multifactor complex of eukaryotic initiation factors, eIF1, eIF2,
eIF3, eIF5, and initiator tRNA

13037–13042.
10. Negrutskii, B.S., Stapulionis, R. & Deutscher, M.P. (1994)
Supramolecular organization of the mammalian translation sys-
tem. Proc.NatlAcad.Sci.USA91, 964–968.
11. Stapulionis, R. & Deutscher, M.P. (1995) A channeled tRNA
cycle during mammalian protein synthesis. Proc. Natl Acad. Sci.
USA 92, 7158–7161.
12. Negrutskii, B.S. & Deutscher, M.P. (1991) Channeling of ami-
noacyl-tRNA for protein synthesis in vivo. Proc. Natl Acad. Sci.
USA 88, 4991–4995.
13. Negrutskii, B.S. & El’skaya, A.V. (1998) Eukaryotic translation
elongation factor 1a: structure, expression, functions, and possible
role in aminoacyl-tRNA channeling. Prog. Nucleic Acids Res.
Mol. Biol. 60, 47–78.
14. Negrutskii, B.S., Budkevich, T.V., Shalak, V.F., Turkovskaya,
G.V. & El’skaya, A.V. (1996) Rabbit translation elongation factor
1a stimulates the activity of homologous aminoacyl-tRNA syn-
thetase. FEBS Lett. 382, 18–20.
15. Reed, V.S., Wastney, M.E. & Yang, D.S.H. (1994) Mechan-
isms of the transfer of aminoacyl-tRNA from aminoacyl-tRNA
synthetase to the elongation factor 1a. J. Biol. Chem. 269, 3293–
3296.
16. Petrushenko, Z.M., Negrutskii, B.S., Ladokhin, A.S., Budkevich,
T.V., Shalak, V.F. & El’skaya, A.V. (1997) Evidence for the for-
mation of an unusual ternary complex of rabbit liver EF-1a with
GDP and deacylated tRNA. FEBS Lett. 407, 13–17.
17.Rether,B.,Bonnet,J.&Ebel,J.P.(1974)StudiesontRNA
nucleotidyltransferase from baker’s yeast. 1. Purification of the
enzyme. Protection against thermal inactivation and inhibition by
several substrates. Eur. J. Biochem. 50, 281–288.

26. El’skaya, A.V., Ovcharenko, G.V., Palchevskii, S.S., Petrushenko,
Z.M., Triano-Alonso, F.J. & Nierhaus, K.H. (1997) Three tRNA
binding sites in rabbit liver ribosomes and role of the intrinsic
ATPase in 80S ribosomes from higher eukaryotes. Biochemistry
36, 10492–10497.
27. Bilgin, N., Ehrenberg, M., Ebel, C., Zaccai, G., Sayers, Z., Koch,
M.H.,Svergun,D.I.,Barberato,C.,Volkov,V.,Nissen,P.&
Nyborg, J. (1998) Solution structure of the ternary complex
between aminoacyl-tRNA, elongation factor Tu and guanosine
triphosphate. Biochemistry 37, 8163–8172.
28. Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshet-
nikova, L., Clark, B.F.C. & Nyborg, J. (1995) Crystal structure of
the ternary complex of Phe-tRNA
Phe
,EF-Tu,andaGTPanalog.
Science 270, 1464–1472.
29. Sioud, M. & Jespersen, L. (1996) Enhancement of hammerhead
ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase.
J. Mol. Biol. 257, 775–789.
30. Dell, V.A., Miller, D.L. & Johnson, A.E. (1990) Effects of
nucleotide- and aurodox-induced changes in elongation factor Tu
conformation upon its interactions with aminoacyl transfer RNA.
A fluorescence study. Biochemistry 29, 1757–1763.
31. Canziani, O., Zhang, W., Cines, D., Rux, A., Willis, B.,
Cohen, G., Eisenberg, R. & Chaiken, I. (1999) Exploring bio-
molecular recognition using optical biosensors. Methods 19,253–
269.
32. Andersen, G.R., Pedersen, L., Valente, L., Chatterjee, I., Kinzy,
T.G., Kjeldgaard, M. & Nyborg, J. (2000) Structural basis for
nucleotide exchange and competition with tRNA in the yeast