From the
The elongation factor Tu (EF-Tu) is a member of the
GTP/GDP-binding proteins and interacts with various partners during the
elongation cycle of protein biosynthesis thereby mediating the correct
binding of aminoacylated transfer RNA (aa-tRNA) to the acceptor site
(A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its
GDP-bound state. In vivo, EF-Tu is post-translationally
modified by phosphorylation. Here we report that the phosphorylation of
EF-Tu by a ribosome associated kinase activity is drastically enhanced
by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function,
inhibits the modification. This effect is specific, since
kirromycin-resistant mutants do become phosphorylated in the presence
of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu
does not bind kirromycin. Most interestingly, the phosphorylation of
EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation
the site of modification is located at the interface between domains 1
and 3 and is involved in a strong interdomain hydrogen bond.
Introduction of a charged phosphate group at this position will change
the interaction between the domains, leading to an opening of the
molecule reminiscent of the GDP conformation. A model for the function
of EF-Tu phosphorylation in protein biosynthesis is presented.
The bacterial peptide chain elongation factor Tu belongs to the
guanosine triphosphatase superfamily of proteins which act as molecular
switches with active and inactive forms depending on the
phosphorylation state of the bound guanine nucleotide (Bourne et
al., 1991). In prokaryotes, EF-Tu
Protein
biosynthesis is the target of numerous antibiotics (Nierhaus, 1982).
The antibiotic kirromycin binds with high affinity to EF-Tu forcing
this protein into a conformation that is unable to maintain its proper
function during the translational elongation cycle. The affinity of the
EF-Tu
It is
known of the E. coli EF-Tu that the protein is N terminally
acetylated (Arai et al., 1980; Jones et al., 1980)
and that lysine 56 can be mono- or dimethylated in vivo (L'Italien and Laursen, 1979). Recently we have shown that
EF-Tu from E. coli and Thermus thermophilus are
post-translationally modified by phosphorylation in vivo (Lippmann et al., 1991, 1993). Phosphorylation of the
eukaryotic EF-1
Standard chemicals were purchased from Merck (Darmstadt,
Germany) and were of the highest quality available. Radiochemicals and
x-ray films were obtained from Amersham (Braunschweig, Germany).
Kirromycin was a gift from A. Parmeggiani (Palaiseau, France), other
antibiotics were from Serva (Heidelberg, Germany) and nucleotides from
Boehringer (Mannheim, Germany). PhastGels and buffer strips were
purchased from Pharmacia (Freiburg, Germany). EF-Tu
To investigate the influence of different substances on EF-Tu
phosphorylation and the binding properties of the modified factor, we
established a simplified in vitro system as described under
``Materials and Methods.''
In short, cells were disrupted
by sonification, the lysate cleared by centrifugation (S15) and in some
experiments the S15 separated into crude 70 S ribosomes (P100) and
supernatant fraction (S100) by a short high-speed centrifugation. The
assays consisted of freshly prepared subcellular fractions (S15, P100,
S100) as source of the kinase and addition of
[
We used another method to analyze the ternary complex
formation. After phosphorylation of EF-Tu the samples were desalted,
GDP was converted to GTP by phosphoenolpyruvate and pyruvate kinase in
the presence of aa-tRNA as described under ``Materials and
Methods.'' Samples were then separated by native gel
electrophoresis in the presence of GTP. Under these conditions EF-Tu
was able to form a ternary complex (Fig. 4, left) but
autoradiography revealed the absence of the phosphorylated part in the
complex (Fig. 4, right). Due to additional steps the
degree of phosphorylation decreased because the phosphatase was still
present in the preparation until the electrophoresis.
The data
on the influence of kirromycin on the phosphorylation and the inability
of the modified EF-Tu to interact with the antibiotic suggest a
specific step in protein biosynthesis as function for the in vivo phosphorylation. Kirromycin prevents the release of EF-Tu
The stimulation of EF-Tu phosphorylation by EF-Ts, another
component of the translational machinery, strongly supports a function
of this post-translational modification in protein biosynthesis. The
interaction between EF-Tu and EF-Ts could lead to a structural
rearrangement of EF-Tu facilitating the access of the kinase to the
site of phosphorylation. Inhibition of the phosphorylation by
kirromycin is also a direct EF-Tu-correlated effect and not mediated by
inhibition of the kinase as can be concluded from experiments in which
EF-TuA
We have attempted to analyze whether EF-Tu is accessible for the
kinase in the ternary complex. The analysis was complicated by the fact
that another kinase has to be added to the system to ensure complete
conversion of GDP to GTP. Therefore we used the nucleotide-free EF-Tu
from T. thermophilus and complexed it with GTP and aa-tRNA
(data not shown). The presence of aa-tRNA reduced the phosphorylation
slightly as compared to GTP alone (in Fig. 7, last
lane), but after addition of an substantial amount of EF-Ts which
ensures complete ternary complex formation no phosphorylated EF-Tu
could be observed after 15 min reaction (data not shown). This led us
to the assumption that in the ternary complex EF-Tu is not accessible
for the kinase. In a complementary way, phosphorylated EF-Tu is not
able to bind aa-tRNA.
Only limited knowledge exists about the
EF-Tu
The inhibition of phosphorylation by kirromycin
implies an antagonistic conformational change. On the other hand, the
antibiotic can be present in complexes of EF-Tu with GDP, GTP, and
aa-tRNA, but not in complex with phosphorylated EF-Tu. This suggests
that the modified factor adopts a new conformation different from the
GDP/GTP/aa-tRNA complexed form. This may occur after the GTP hydrolysis
when EF-Tu
The inability of the phosphorylated EF-Tu to bind
aa-tRNA points to a similar behavior on the ribosome and suggests the
following model. After binding of the ternary complex to the A-site of
the ribosome the EF-Tu affinity to aa-tRNA is changed by GTP
hydrolysis, which opens the EF-Tu complex for the kinase. The
phosphorylation will then loosen the binding of EF-Tu to the ribosomal
complex, thus facilitating EF-Tu to leave the site of translation.
As stated in the first report on the phosphorylation of EF-Tu
(Lippmann et al., 1993) the degree of phosphorylation in
vivo is low, approximately 5%. In our in vitro studies we
reached a higher rate only with the enhancement by EF-Ts. Preliminary
experiments with a partially purified kinase activity did not
considerably increase the extent of phosphorylation. This suggests that
the limiting step in phosphorylation is not the kinase itself but the
substrate. The degree of in vivo modified EF-Tu is in good
correlation with the fraction which is active on the ribosome at a
given time point.
Assuming that the affinity of EF-Tu from E.
coli for GDP is 100-fold higher than for GTP and that some GTP is
still present in the S15 preparation used for the in vitro experiments this number of about 5% within the experimental error
also correlates with the fraction initially complexed to GTP.
Saturating amounts of GTP could not be used in the assay because, as
stated above, the kinase can use GTP as well as ATP as substrate and
therefore the specific activity drops and the analysis will be impaired
by a loss of the signal.
These in vivo and in vitro data let us conclude that the fraction of EF-Tu which changes its
conformation from GTP to GDP may be the substrate for the kinase. This
is substantiated by the experiment in which the time course of
phosphorylation of EF-Tu
A comparison of the three-dimensional structures of
the complexes of EF-Tu with GDP (Kjeldgaard and Nyborg, 1992) and with
the GTP analogue, Gpp(NH)p (Berchtold et al., 1993; Kjeldgaard et al., 1993) supports this view. Upon transition from the
active GTP form to the inactive GDP complex, the phosphorylation site
changes position by as much as 21.8 Å (see purple balls in Fig. 8). In the active GTP form, Thr-382 is located
almost inaccessible, in the interface between domain 1 and 3 of EF-Tu;
its side chain hydroxyl group donates a 2.70-Å hydrogen bond to
Glu-117, across this interface (Fig. 9). It is thought that the
aa-tRNA will bind to the cleft identified between domain 1 and 2
(Berchtold et al., 1993; Förster et al., 1993),
possibly locating the aminoacylated CCA-end close to the
phosphorylation site. During binding of the ternary complex to the
ribosome conformational changes of components occur (Rodnina et
al., 1993), possibly of the aa-tRNA after correct alignment to the
codon (Bertram et al., 1983) or of EF-Tu. One or both events
may weaken the interaction between domains 1 and 3, making the site of
phosphorylation accessible for the kinase. Introduction of the
negatively charged phosphate group will lead to the repulsion of
Glu-117 and result in complete disruption of the interface. Such a
mechanism could accelerate the switch to the ``open'' GDP
form, which falls off from the ribosome.
In any case, the
phosphorylation of EF-Tu appears to be a novel mechanism for a
reversible modification of the interaction behavior of the factor and
thus for the regulation of protein synthesis in general.
We thank H. Ebeling for photography and W. Rossner for
carefully reading the manuscript. The generous gifts of kirromycin from
A. Parmeggiani and of EF-Ts from S. Lorenz are gratefully acknowledged.
We thank M. Sprinzl for providing us nucleotide-free EF-Tu and for
fruitful discussions and critical comments.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)is the
most abundant protein. The active EF-Tu
GTP complex catalyzes the
binding of aminoacyl-tRNA to the programmed ribosome. Activity is
terminated after the hydrolysis of guanosine triphosphate (GTP), and
the resulting guanosine diphosphate (GDP) has to be replaced by GTP in
the elongation factor Ts-mediated guanine-nucleotide exchange reaction.
EF-Ts is an essential protein for the growth of Escherichia coli and its expression is tightly regulated (Hwang et al.,
1989) but only little is known about the molecular mechanism of its
interaction with EF-Tu (Hwang et al., 1992).
GTP
kirromycin complex toward aa-tRNA is drastically
reduced (Abrahams et al., 1991) while even the GDP form of
EF-Tu promotes binding of aa-tRNA to the ribosomal A-site in presence
of the antibiotic (Wolf et al., 1974). The inhibitory effect
of kirromycin on translation is due to the fact that the release of
EF-Tu
GDP from the acceptor site of the ribosome is prevented
(Wolf et al., 1977). Crystallographic studies have shown that
EF-Tu consists of three distinct domains: domain 1 (residues
1-200), domain 2 (residues 209-299), and domain 3 (residues
300-393) (Kjeldgaard and Nyborg, 1992; Berchtold et al.,
1993; Kjeldgaard et al., 1993). Analysis of
kirromycin-resistant mutants (Zeef and Bosch, 1993) revealed that the
antibiotic may interact at the interface of domains 1 and 3 when EF-Tu
is in the GTP bound state (Mesters et al., 1994).
(the eukaryotic counterpart of EF-Tu) was also
described, but the site of modification was not determined (Venema et al., 1991a, 1991b). We identified the site of
phosphorylation in E. coli EF-Tu as threonine 382 (Lippmann et al., 1993). This residue is strictly conserved in all known
EF-Tu and EF-1
sequences. The modification can be reversed by the
action of a phosphatase. Here we report the influence of the
phosphorylation on the interaction of EF-Tu with EF-Ts, kirromycin, and
aa-tRNA.
GDP from E. coli was isolated as described by Leberman et al. (1980) and further purified by crystallization (Lippmann et
al., 1988).
In Vitro Phosphorylation Assay
In vitro phosphorylation of EF-Tu was performed in a simplified assay
system using cleared whole cell lysate (S15) as source of
EF-Tu-specific protein kinase activity. The S15 results in the same
phosphorylation effect as the crude 70 S ribosomes preparations
described (Lippmann et al., 1993). Frozen cells from mid-log
phase (E. coli MRE 600) where resuspended in assay buffer (50
mM TrisHCl, pH 7.4, 10 mM MgCl
, 15
mM dithioerythritol, 1 mM phenylmethylsulfonyl
fluoride) and lysed by sonification. Cell debris were removed by
centrifugation (15,000
g, 20 min, 4 °C) and the
absorption at 260 nm of the supernatant (``S15'') was
determined. In vitro phosphorylation assays (final volume 10
µl) were performed in the same buffer containing 5 µM
EF-Tu, 150 µM [
-
P]ATP
(specific activity 250 GBq mmol
), and 0.01 (or 0.25) A
of S15 extract at 30 °C. Additional
components tested for influence on EF-Tu phosphorylation were present
as indicated in the corresponding figures. Reactions were terminated by
addition of sample buffer (Laemmli) and boiling for 5 min and subjected
to SDS-PAGE (12%). For removal of nucleic acids (RNA + DNA), the
gels were treated with 7% (v/v) trichloroacetic acid (30 min, 95
°C) prior to Coomassie staining. After drying, gels were exposed to
x-ray films.
Native Gel Electrophoresis with the
PhastSystem
Native buffer strips for the PhastSystem (Pharmacia)
were equilibrated in 8 mM TricineNaOH, pH 8.2, for more
than 1 h. Separation was performed in 20% homogeneous polyacrylamide
PhastGel in a PhastSystem (Pharmacia) under the following running
conditions. Sample application at step 2 from 0 V-h (down) to 2 V-h
(up); separation step 1: 400 V, 10 mA, 2.5 W at 4 °C for 10 V-h;
separation step 2: 400 V, 1 mA, 2.5 W at 4 °C for 2 V-h; separation
step 3: 400 V, 10 mA, 2.5 W at 4 °C for 288 V-h. Note that only one
gel could be run at a time because two gels exceeded the cooling
capacity of the system.
Native Electrophoresis in the Presence of
GTP
After phosphorylation of EF-Tu by S15, samples were desalted
and converted to EF-TuGTP. In the presence of 100 pmol of EF-Tu,
tRNA
was added (as indicated in Fig. 6) in a
50-µl reaction volume in polymix buffer (5 mM Mg-acetate,
0.5 mM CaCl
, 95 mM potassium chloride, 5
mM NH
Cl, 8 mM putrescine, 1 mM
spermidine, 5 mM potassium phosphate, 1 mM dithioerythritol, pH 7.5) containing 1 mM ATP, 10
mM phosphoenolpyruvate, 1 mM GTP, 5 µg of
pyruvate kinase, 0.3 µg of myokinase, and 10 units of Phe-tRNA
synthetase. Complex formation was at 37 °C for 10 min. Samples were
cooled on ice and 0.1 volume of 50% glycerol was added. Samples were
separated on 5% acrylamide gels at 4 °C with circulation of the
buffer (50 mM Tris
HCl, 10 mM Mg-acetate, 65
mM NH
acetate, 1 mM EDTA, 1 mM
dithioerythritol, 10 µM GTP, pH 6.8).
Figure 6:
Kirromycin resistance overcomes the
phosphorylation inhibition of the antibiotic. Analysis of EF-Tu
phosphorylation by different S15 preparations was performed in the
absence (-) or presence (+) of 50 µM
kirromycin. Upper panel, phosphorylation analysis was
performed with 0.25 A S15 from the mentioned
strain without addition of EF-Tu, using the endogenous EF-Tu as
substrate. As could be inferred from the figure, the antibiotic
efficiently blocks phosphorylation in the wild-type strain MRE 600, but
did not show any effect on the modification of mutant EF-TuA
from the PM455 strain. Lower panel, catalytic amounts of
S15 from the strains indicated were analyzed for their ability to
phosphorylate wild-type EF-Tu. The kinase of the mutant strain is not
able to modify the wild-type EF-Tu. A, displays Coomassie
staining; B, autoradiography.
-
P]ATP and EF-Tu as substrates. The extent
of modification was monitored by SDS-PAGE followed by autoradiography.
In this system we analyzed the effect of EF-Ts. Addition of EF-Ts
resulted in an increase in EF-Tu phosphorylation, which was most
significant in the crude ribosomal fraction (Fig. 1, A and B). Neither EF-Ts nor EF-Tu, nor the combination of
both, displayed any incorporation of radioactivity in the absence of
S15, excluding EF-Ts itself as the kinase. The stoichiometry of the
EF-Ts activation was monitored with S15. Maximal enhancement was
reached at an EF-Ts concentration nearly equimolar to that of EF-Tu (Fig. 1C). Apparently, interaction with EF-Ts induces a
conformational change in EF-Tu exposing the phosphorylation site to the
kinase.
Figure 1:
Exchange factor EF-Ts enhances EF-Tu
phosphorylation. Crude cellular fractions were prepared as described
elsewhere (Lippmann et al., 1993). From each fraction 0.01 A aliquots were analyzed for their ability to
phosphorylate EF-Tu in the absence or presence of EF-Ts in an equimolar
concentration to EF-Tu. Samples were separated on 12% SDS-PAGE. The
enhancement of the phosphorylation by EF-Ts is manifold stronger in the
crude 70 S ribosome preparation than in S15, indicating an association
of the kinase activity with these translational components. Addition of
EF-Ts to EF-Tu alone in the presence of
[
-
P]ATP leads to no incorporation of
radioactivity into EF-Tu (right lane). A, Coomassie Blue
staining of the SDS-PAGE; B, autoradiography. C,
concentrations of EF-Tu (5.4 µM) and S15 (0.01 A
) were kept constant and increasing amounts of
EF-Ts were added. Quantifications were done by densitometry of the
autoradiogram (inset), corrected by estimation of protein
quantities from densitometry of the Coomassie-stained
gel.
We further examined the influence of various antibiotics
acting on protein biosynthesis in the in vitro phosphorylation
system. Using S15 as source for kinase, the presence of 20- to 100-fold
excess over EF-Tu of puromycin, chloramphenicol, or streptomycin
resulted neither in significant enhancement nor in reduction of EF-Tu
modification (data not shown), but kirromycin abolished the
phosphorylation completely (Fig. 2).
Figure 2:
Inhibition of EF-Tu phosphorylation by
kirromycin. Wild-type EF-TuGDP from E. coli was
incubated for 20 min with catalytic amounts (0.01 A
) of S15 from E. coli MRE 600 in the
presence of [
-
P]ATP and separated by
SDS-PAGE as described under ``Materials and Methods.'' The inset shows an example (molecular mass from the marker are 94,
67, 43, and 29 kDa). The graph displays data derived from densitometry
of autoradiograms, corrected for protein content by densitometry of the
Coomassie-stained gel. The dotted line indicates the EF-Tu
concentration under assay conditions.
Therefore, we analyzed
the influence of this antibiotic on EF-Tu modification in more detail.
Addition of kirromycin in increasing concentrations led to a sharp
reduction in phosphorylation under conditions of 1:1 binding (Fig. 2). To clarify whether the inhibition is an effect of
binding to EF-Tu and not due to a dephosphorylation by kirromycin (or
stimulation of a highly active phosphatase) we used a native gel
electrophoresis system under nonequilibrium conditions using
commercially available mini gels (PhastGel; Pharmacia). After
optimizing the buffer and running conditions EF-Tukirromycin
complexes could be separated from free EF-Tu. Results are shown in Fig. 3, upper panel, lane 4. As can be
concluded from the Coomassie staining, the nonphosphorylated EF-Tu
displayed normal kirromycin binding properties, leading to a higher
migration velocity than the free EF-Tu, whereas the phosphorylated
portion did not participate in this interaction, migrating to the same
position as the control (Fig. 3, lane 1). From the
intensity of Coomassie staining of the nonphosphorylated complexed
EF-Tu and the free phosphorylated protein the extent of modification
was estimated to approximately 7% (lane 4). This is the first
demonstration of a wild-type form of EF-Tu that is unable to recognize
kirromycin.
Figure 3:
Phosphorylated EF-Tu is unable to bind
kirromycin, but is still capable to complex EF-Ts. Complexes were
formed at 0 °C after phosphorylation of EF-Tu by S15 extract and
separated by native PAGE (see ``Materials and Methods'') on
20% homogeneous PhastGels as shown in the upper panel and
SDS-PAGE (12%; lower panel). Lane 1, without any
addition; lane 2, addition of EF-Ts (1:1 molar ratio to
EF-Tu); lane 3, addition of EF-Ts and kirromycin (1:2:2 molar
ratio of EF-Tu:EF-Ts:kirromycin); lane 4, addition of 10-fold
molar excess of kirromycin (over EF-Tu); lane 5, addition of
5-fold molar excess of EF-Ts (over EF-Tu). Note: in lane 5 the
running behavior of free EF-Ts is displayed. The EF-Ts migrates as a
doublet (only under native conditions; compare upper and lower panel), the upper band may indicate a dimer. Both bands
disappeared in the presence of saturating amounts of EF-Tu, confirming
the identity as EF-Ts (lane 2). The phosphorylated EF-Tu is
clearly capable of binding to EF-Ts (lanes 2, 3, and 5), but not to kirromycin (lanes 3 and 4).
The native gel system enabled us to investigate also the
interaction behavior of the modified elongation factor Tu with its
exchange factor EF-Ts. In these experiments the ability of the
phosphorylated EF-Tu to complex EF-Ts could be demonstrated (Fig. 3, lanes 2, 3, and 5). The phosphorylated
part seems to have a higher affinity for EF-Ts as can be concluded from
data where EF-Tu was in excess over EF-Ts (not shown). Under the
running conditions employed, free EF-Ts migrated as two separate bands (Fig. 3, lane 5) which both were complexed in the
presence of saturating amounts of EF-Tu (Fig. 3, lane
2).
Figure 4:
Phosphorylated EF-Tu is unable to bind
aa-tRNA. After phosphorylation of EF-Tu by S15 extract samples were
desalted and converted to EF-TuGTP as described under
``Materials and Methods.'' Analysis of 100 pmol of EF-Tu for
aa-tRNA binding was performed in a 5% PAGE under native conditions with
circulating buffer. After fixation in 5% acetic acid and 50% methanol
the gel was boiled in 5% trichloroacetic acid for 30 min to remove all
nucleic acids. As displayed by Coomassie staining (left) the
EF-Tu is able to bind aa-tRNA, but on the autoradiogram (right) no phosphorylated EF-Tu could be detected at the
position of the ternary complex.
To further
analyze the inability of the post-translationally modified EF-Tu to
bind kirromycin and aa-tRNA, we employed a method which is especially
suitable to detect weak interactions under equilibrium conditions,
namely zone-interference gel electrophoresis (Abrahams et al.,
1988). The result confirmed the absence of phosphorylated EF-Tu in
complex with kirromycin and aa-tRNA (Fig. 5). From the ligand
concentration in the zones one can deduce, that the K values for eventual EF-Tu complexes
with kirromycin and aa-tRNA should at least be higher than 25
µM.
Figure 5:
EF-Tu phosphorylation even prevents weak
interactions with kirromycin and aa-tRNA. Zone-interference gel
electrophoresis (Abrahams et al., 1988) was applied to examine
the binding of phosphorylated EF-Tu (pEF-Tu) to kirromycin. An
EF-Tu preparation phosphorylated by S15 as described under
``Materials and Methods'' was analyzed, using samples and
zones with concentrations of kirromycin and aa-tRNA as indicated. The
preparation displayed a normal binding of kirromycin and aa-tRNA,
except the phosphorylated part which did not show any interaction
migrating to the same position as the control. Panel A,
Coomassie Blue staining; panel B,
autoradiography.
In the three-dimensional structure the mutation
A375T leading to kirromycin resistance is close to the site of
phosphorylation (Kjeldgaard and Nyborg, 1992; Berchtold et
al., 1993; Mesters et al., 1994). The
kirromycin-resistant EF-Tu mutants are able to promote polypeptide
chain growth even in the presence of high concentrations of the
antibiotic, whereas in wild-type cells EF-TuGDP
kirromycin
sticks to the ribosome. Because kirromycin also blocks phosphorylation
we investigated the modification of a mutated EF-Tu. We used the E.
coli strain PM455 carrying a mutant tufA gene coding for
kirromycin-resistant EF-TuA
(EF-Tu(Ala
-Thr))
and an inactivated tufB gene (Mu insertion) (Van der Meide et al., 1982). As displayed in Fig. 6(upper
panel), the mutant EF-TuA
was modified to the same
extent both in the presence and absence of kirromycin and showed an
even stronger degree of phosphorylation than wild-type EF-Tu of an
equally prepared S15 extract from E. coli MRE 600.
Surprisingly, catalytic amounts of the S15 preparation from the mutant
strain appeared to be unable to mediate phosphorylation of wild-type
EF-Tu (Fig. 6, lower panel). Identical results were
obtained with another strain (LZ13; Zeef and Bosch, 1993) carrying the
same mutations concerning the EF-Tu genes (data not shown).
GDP
from the ribosome after GTP hydrolysis. To analyze whether this is the
point where phosphorylation occurs, we performed the following
experiment. Nucleotide-free EF-Tu was saturated with GTP and used as
substrate in the phosphorylation assay. Because the nucleotide-free
form of E. coli EF-Tu is hard to prepare we changed to T. thermophilus. S15 was also prepared from T. thermophilus as described for E. coli.
The phosphorylation reaction was studied at 50 °C as a function of
time. Under these conditions one may expect that the EF-Tu
GTP
will rapidly be converted into EF-Tu
GDP (Limmer et al.,
1992). The result is shown in Fig. 7. At the time points
indicated aliquots were removed and analyzed on SDS-PAGE followed by
autoradiography. One can see that at the beginning where the
ribosome-stimulated transition rate from GTP to GDP complex is fast,
the degree of phosphorylation is also enhanced. In addition, the
experiment shows that the reaction is reversible. Despite the excess of
ATP and the permanent presence of EF-Ts, the degree of phosphorylation
diminishes when the amount of GTP becomes exhausted.
Figure 7:
Time course of phosphorylation of
EF-TuGTP. Nucleotide-free EF-Tu from T. thermophilus HB8 was saturated by a 4-fold molar excess (300 µM)
of GTP prior to phosphorylation by S15 from T. thermophilus HB8 prepared essentially as described for E.
coli. The reaction was started by addition of S15 and incubation
at 50 °C. At the time points indicated aliquots were removed and
the reaction terminated by boiling with sample buffer. Only the
relevant portion of the autoradiogram is
shown.
In summary, we
could demonstrate that phosphorylation of EF-Tu can be inhibited by
kirromycin and enhanced by EF-Ts. The modification abolishes the
complex formation between EF-Tu and kirromycin as well as aa-tRNA.
Kirromycin-resistant cells display a recognition between EF-Tu and
kinase different from that in the case of sensitive cells. The
reversible phosphorylation of EF-Tu is correlated with the transition
from EF-TuGTP to EF-Tu
GDP.
was phosphorylated by wild-type S15 (MRE 600) in the
presence of kirromycin (data not shown). The inability of the mutant
strain kinase to modify the wild-type EF-Tu underlines the importance
of the phosphorylation for proper function. The small structural
aberration by the single amino acid replacement in EF-TuA
seems to be compensated by a parallel mutation in the kinase.
EF-Ts interaction. Binding experiments suggests involvement
of domains 2 and 3 of EF-Tu (Peter et al., 1990), whereas
mutant analysis refer to domain 1 (Hwang et al., 1992). Recent
results point to an extended EF-Ts interaction during the activity
cycle of EF-Tu, going from nucleotide exchange up to a quaternary
complex of EF-Tu
GTP
aa-tRNA
EF-Ts that enters the
ribosome (Bubunenko et al., 1992; Schwartzbach and Spremulli,
1991). Ehrenberg and co-workers (Ehrenberg et al., 1990;
Scoble et al., 1994) and Weijland and Parmeggiani(1993) have
shown that 2 molecules of GTP are hydrolyzed per peptide bond formation
by the action of EF-Tu. The presence of EF-Ts in a complex on the
translating ribosome would allow a direct exchange of the GDP
hydrolyzed. We have found that the major kinase activity is associated
with a crude 70 S preparation (Lippmann et al.(1993) and Fig. 1) and that EF-Tu phosphorylation is enhanced by the
addition of EF-Ts.
GDP(
EF-Ts) is still on or close to the
translating ribosome. Although the kinase can use GTP as well as ATP as
substrate we can rule out that the
-phosphate of the GTP complexed
to EF-Tu is the source for phosphorylation.
(
)
GTP was followed. Under the conditions
employed the transition rate from GTP to GDP complex is initially high
until the majority of the GTP is hydrolyzed. The result (Fig. 7)
clearly demonstrated that this is in correlation with the degree of
phosphorylation.
Figure 8:
Structure of EF-TuGDP and
EF-Tu
Gpp(NH)p. Ribbon model of EF-Tu in complex with GDP (top) and with Gpp(NH)p (bottom). Domain 1 is shown
in orange, domain 2 in yellow, and domain 3 in blue. The nucleotide in
domain 1 is displayed in green and the white sphere indicates the
Mg
ion. The site of phosphorylation is indicated by a
purple sphere in domain 3. The switch between the two states moves the
phosphorylation site by approximately 21
Å.
Figure 9:
The
phosphorylation site of EF-Tu in the structure of the Gpp(NH)p complex.
Stereo illustration of the interaction between domain 1 (orange ribbon)
and domain 3 (blue ribbon) around the site of phosphorylation. Residues
are numbered according to E. coli EF-Tu. Dashed lines shows hydrogen bonds, red crosses represent positions of
water molecules.
The inhibition of
phosphorylation of Thr-382 by kirromycin is also in agreement with the
structural data. Mesters et al.(1994) have shown that the
presumable binding site for kirromycin on the E. coli EF-Tu is
along the interface between domains 1 and 3, since all the known
resistance mutations are located here. Two of these, Gln-124 and
Thr-375, are spatially close to Thr-382 (see Fig. 9). Thus, it
may be that the inhibition of phosphorylation by the antibiotic
provides an explanation for the immobilization of
EF-TuGDP
kirromycin on the ribosome.
,
-imino)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.