Originally published In Press as doi:10.1074/jbc.M002496200 on April 25, 2000
J. Biol. Chem., Vol. 275, Issue 27, 20458-20466, July 7, 2000
Kinetics of the Interaction of Translation Factor SelB from
Escherichia coli with Guanosine Nucleotides and
Selenocysteine Insertion Sequence RNA*
Martin
Thanbichler
,
August
Böck, and
Roger S.
Goody§¶
From the Lehrstuhl für Mikrobiologie der
Universität München, Maria-Ward-Straße 1a, 80638 München, Germany and the § Max-Planck-Institut
für Molekulare Physiologie, Rheinlanddamm 201, 44026 Dortmund, Germany
Received for publication, March 24, 2000
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ABSTRACT |
The kinetics of the interaction of GTP and GDP
with SelB, the specific translation factor for the incorporation of
selenocysteine into proteins, have been investigated using the
stopped-flow method. Useful signals were obtained using intrinsic
(i.e. tryptophan) fluorescence, the fluorescence of
methylanthraniloyl derivatives of nucleotides, or fluorescence
resonance energy transfer from tryptophan to the methylanthraniloyl
group. The affinities of SelB for GTP (Kd = 0.74 µM) and GDP (Kd = 13.4 µM) were considerably lower than those of other
translation factors. Of functional significance is the fact that the
rate constant for GDP release from its complex with SelB (15 s
1) is many orders of magnitude larger than
for elongation factor Tu, explaining why a GDP/GTP exchange factor is
not required for the action of SelB. In contrast, the rate of release
of GTP is 2 orders of magnitude slower and not significantly faster
than for elongation factor Tu. Using a fluorescently labeled
17-nucleotide RNA minihelix that represents a binding site for the
protein and that is part of the fdhF selenocysteine
insertion sequence element positioned immediately downstream of the UGA
triplet coding for selenocysteine incorporation, the kinetics of the
interaction were studied. The high affinity of the interaction
(Kd ~ 1 nM) appeared to be increased
even further when selenocysteyl-tRNASec was bound to SelB,
but to be independent of the presence or nature of the guanosine
nucleotide at the active site. These results suggest that the affinity
of SelB for its RNA binding site is maximized when charged tRNA is
bound and decreases to allow dissociation and reading of codons
downstream of the selenocysteine codon after selenocysteine peptide
bond formation.
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INTRODUCTION |
The specialized translation factor SelB is the key molecule for
the specific incorporation of the amino acid selenocysteine into
polypeptides. SelB binds guanine nucleotides;
selenocysteyl-tRNASec 1; and
a secondary structure of the mRNA, the SECIS element. In bacterial
selenoprotein mRNAs, this SECIS element is located immediately downstream of the UGA codon directing selenocysteine insertion, whereas
in Archaea and Eukarya, it is situated in the 3'-nontranslated region
(for a review, see Ref. 1). Binding of SelB to the SECIS structure is
mediated by an ~17-kDa C-terminal domain of the protein (domain IVb)
that maintains its binding capacity when separated from the rest of the
molecule. Minimization of the SECIS element showed that a 17-nucleotide
long minihelix (see Fig. 1) was still able to bind to SelB or its
C-terminal domain and to promote selenocysteine incorporation in
vivo. On the other hand, the N-terminal two-thirds of SelB, which
display sequence similarity to elongation factor Tu, are still capable
of binding selenocysteyl-tRNASec when separated from the
mRNA binding domain (2, 3).
The formation of the quaternary complex is essential for the decoding
of the UGA codon with selenocysteine at the ribosome. It is assumed
that within this complex, SelB attains a conformation rendering it
compatible for interaction with the ribosome, which is required for
triggering GTPase activity. It is further assumed that GTP hydrolysis
changes the conformation such that the charged tRNA is released.
Therefore, in this model, SelB has two functions: the first one is to
discriminate between an UGA codon programmed for selenocysteine
insertion by the SECIS element and an ordinary UGA stop codon that
lacks the SECIS element (4). Furthermore, in bacterial mRNAs coding
for selenocysteine, SelB also tethers the tRNA to the ribosomal A site
(4, 5).
Characterization of the kinetics of the SelB interaction with its
substrates and the conformational changes involved is therefore of
paramount importance for understanding the decoding process. Equilibrium dialysis measurements of the interaction of the protein with guanine nucleotides had indicated that GDP binding is
approximately an order of magnitude weaker that GTP binding (6). This
could obviate the necessity for a guanine nucleotide release factor. It
has also been observed that SelB forms a tighter complex with the SECIS
element in the presence of selenocysteyl-tRNASec than in
its absence (7). This study deals with a detailed analysis of the
association and dissociation kinetics of guanine nucleotides with SelB
alone and in the presence of the mRNA. Additionally, the kinetics
of the interaction of SelB with SECIS elements and the influence of
guanine nucleotides and selenocysteyl-tRNASec thereon were investigated.
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EXPERIMENTAL PROCEDURES |
Materials--
L-[14C]Serine was
obtained from NEN Life Science Products and had a specific activity of
160 mCi/mmol. Sodium [75Se]selenite with a specific
activity of 296 mCi/mmol and RNase P1 were purchased from Amersham
Pharmacia Biotech (Freiburg, Germany). Yeast inorganic pyrophosphatase
was from Sigma-Aldrich (Deisenhofen, Germany). Bovine serum albumin and
bakers' yeast tRNA were obtained from Roche Molecular Biochemicals
(Mannheim, Germany). The 17-nucleotide long minihelix representing the
binding part of the SECIS element of the fdhF mRNA from
Escherichia coli (see Fig. 1) was prepared as described
previously (2) and was a generous gift from M. Kromayer. The labeled
fdhF minihelix carrying a
(3',6'-dipivaloylfluoresceinyl)-6-carboxamidohexyl group at the 5'-end
(5'-fluorescein-GGUUGCAGGUCUGCACC-3') was synthesized by Interactiva
(Ulm, Germany). Selenocysteine synthase (SelA) purified as described
(8) was a generous gift from K. Forchhammer. T7 RNA polymerase was
isolated as described (9) and kindly provided by S. Leonhartsberger.
Purification of SelB and SelB-(472-614)--
For purification
of SelB, E. coli strain BL21(DE3) (10) was transformed with
plasmid pWL194 (11) harboring selB under control of the T7
10-promoter. Transformants were grown in a 10- or 50-liter Biostat
fermentor (B. Braun, Dassel, Germany) at 37 °C in a medium
containing 3% peptone, 1% yeast extract, 0.4% glucose, 0.2%
glycerol, 25 mM NaCl, 2 mM MgCl2,
60 mM potassium phosphate (pH 7.1), and 100 µg/ml
ampicillin. At an A600 of 3.0, expression of
selB was induced by addition of 100 µM
isopropyl-
-thiogalactopyranoside. Subsequently, the temperature was
lowered to 30 °C. After 75-150 min, the suspension was chilled to
0 °C, and the cells were harvested in a continuous flow centrifuge.
Breakage of the cells, removal of cell debris, enrichment of SelB by
sedimentation with the ribosomes and subsequent extraction from the
pellet with high salt buffer, and fractionated ammonium sulfate
precipitation were performed as described (11). The final precipitate
was dissolved in 50 ml of buffer A (100 mM potassium phosphate (pH 7.0), 2 mM magnesium acetate, 2 mM DTT, and 0.5 mM EDTA). After dialysis for
6 h against two changes (2 liters each) of the same buffer, the
solution was applied to a hydroxylapatite column (1.6 × 12.5 cm;
Bio-Gel HTP, Bio-Rad). The column was developed with 500 ml of a linear
gradient of 100-200 mM potassium phosphate in buffer A at
a flow rate of 0.5 ml/min. SelB was eluted at a potassium phosphate
concentration of ~165 mM. Fractions containing SelB, as
monitored by SDS gel electrophoresis, were pooled and dialyzed against
two changes (2 liters each) of buffer B (100 mM potassium
phosphate (pH 7.0), 5 mM MgCl2, 2 mM DTT, and 0.5 mM EDTA) for 6 and 12 h,
respectively. The pool was then loaded onto a Q-Sepharose
anion-exchange column (1.2 × 8 cm; Amersham Pharmacia Biotech),
which was developed with 90 ml of a linear gradient of 0-267
mM KCl in buffer B at a flow rate of 1 ml/min. SelB was
eluted at ~125 mM KCl. The fractions containing the
protein were pooled and dialyzed for 12 h against 2 liters of
buffer B. The solution was then applied to an SP-Sepharose Fast Flow
cation-exchange column (1 × 5 cm; Amersham Pharmacia Biotech).
The column was developed with 80 ml of a linear gradient of 0-400
mM KCl in buffer B at a flow rate of 1 ml/min. SelB was
eluted at ~230 mM KCl. Fractions that contained SelB were
pooled and dialyzed for 12 h against 2 liters of buffer B. Finally, the solution was concentrated by dialysis for 12 h
against 500 ml of buffer B containing 50% glycerol and stored at
20 °C.
His-tagged SelB-(472-614) was purified essentially as described by
Kromayer et al. (2). Protein concentrations were determined spectroscopically at 280 nm (
SelB = 81,080 M1 cm1,
SelB-(472-614) = 26,600 M1 cm1)
according to Gill and von Hippel (12).
Isolation of Selenophosphate Synthetase--
Purification of
selenophosphate synthetase (SelD) was performed according to the method
of Ehrenreich et al. (13).
Isolation of Seryl-tRNA Synthetase--
Seryl-tRNA synthetase
was purified essentially as described by Härtlein et
al. (14).
Preparation and Folding of the RNA Stem-Loops--
The
39-nucleotide RNA stem-loop comprising most of the fdnG
SECIS element from E. coli (15, 16) (Fig.
1) was synthesized in vitro
using oligonucleotides as a template (9, 17). The reaction mixture
contained 40 mM Tris-HCl (pH 8.1), 40 mM
MgCl2, 1 mM spermidine, 5 mM DTT,
0.01% Triton X-100, 8% polyethylene glycol 8000, 8 mM
GMP, 2.4 units/ml pyrophosphatase, 20 µg/ml T7 RNA polymerase, and a
0.2 µM concentration of the partially double-stranded DNA
template. After incubation at 37 °C for 3 h, the solution was
extracted with phenol (equilibrated with 100 mM sodium
acetate (pH 4.6)). The transcripts were subsequently precipitated with
ethanol and separated on a 17.5% polyacrylamide gel (38:2 cross-link)
under denaturing conditions. After visualization by UV shadowing, the
transcript with the correct length was cut out and electroeluted from
the gel using a Biotrap® chamber (Schleicher & Schüll, Dassel)
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Fig. 1.
Structures of fdhF and
fdnG SECIS elements. Bases that are protected by
SelB against chemical modification (26) are marked by
triangles. The shaded regions were synthesized
in vitro and used for the experiments reported here.
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To remove salt, which strongly favors the formation of biologically
inactive dimers, both the unlabeled and fluorescein-labeled RNA
oligonucleotides were precipitated by addition of 0.1 volume of sodium
acetate (pH 4.6) and 1 volume of isopropyl alcohol before further use.
After incubation at 20 °C, the precipitate was collected by
centrifugation at 16,000 × g for 45 min, washed with
80% ethanol, dried, and resolubilized in 0.1 mM EDTA.
Folding of the stem-loops was performed at a maximal concentration of
200 µM in a volume of 100 µl. The solution was
incubated for 90 s at 80 °C and then rapidly cooled on ice.
Under these conditions, all RNA oligonucleotides exclusively adopted
the stem-loop conformation.
Determination of the RNA Stem-Loop Concentration--
To
determine their exact concentration, RNA stem-loops were hydrolyzed by
RNase P1 in a reaction mixture containing 30 mM sodium
acetate (pH 5.0), 1.6 mM ZnCl2, and 8 units/ml
RNase P1 in a volume of 100 µl. After incubation for 2 h at
60 °C, the solution was diluted with distilled H2O, and
potassium phosphate (pH 7.0) was added to a final concentration of 50 mM. Subsequently, the absorption of the released nucleotide
monophosphates was measured at 260 nm. From the value obtained, the
oligonucleotide concentration was calculated using the specific
absorption coefficients 260 = 177.2 mM1 cm1
for the fdhF minihelix and 260 = 424.8 mM1 cm1
for the fdnG stem-loop. The underlying specific absorption
coefficients (260) of the nucleotide monophosphates were
15.31 M1
cm1 for AMP, 11.67 mM1 cm1
for GMP, 9.82 mM1
cm1 for UMP, and 7.45 mM1 cm1
for CMP.
Isolation of tRNASec--
tRNASec was
purified from E. coli MC4100 (18) carrying plasmid pCB2013
according to the procedure described by Baron and Böck (19). Its
concentration was determined by aminoacylation with L-[14C]serine under saturating seryl-tRNA
synthetase concentrations. The aminoacylation mixture was pipetted into
250 µl of ice-cold 10% trichloroacetic acid containing 0.1%
L-serine. After addition of 100 µg of bakers' yeast
tRNA, the precipitate was transferred onto glass-fiber filters and
washed four times with 1 ml of 10% trichloroacetic acid containing
0.1% L-serine, five times with 1 ml of 5% trichloroacetic
acid containing 0.05% L-serine, and three times with 1 ml
of 80% ethanol. The filters were dried at 65 °C, and their
radioactivity was determined by liquid scintillation counting.
Aminoacylation of tRNASec--
Aminoacylation of
tRNASec was performed in a reaction mixture containing 100 mM Hepes (pH 7.0), 10 mM KCl, 10 mM
magnesium acetate, 1 mM DTT, 10 mM ATP, 200 µM L-serine, 83 µM
tRNASec, 0.1 mg/ml bovine serum albumin, 2.5 units/ml
pyrophosphatase, and 0.84 mg/ml seryl-tRNA synthetase. After incubation
for 15 min at 37 °C, the reaction was stopped by addition of 0.033 volume of 3 M sodium acetate (pH 4.6) and subsequent
treatment with phenol equilibrated with 100 mM sodium
acetate (pH 4.6). Seryl-tRNASec was precipitated from the
aqueous phase at 20 °C by addition of 0.1 volume of 3 M sodium acetate (pH 4.6) and 1 volume of isopropyl alcohol. The precipitate was collected by centrifugation at 16,000 × g for 30 min, washed with 80% ethanol, dried in
vacuo, and resolubilized in 10 mM sodium acetate (pH
4.6).
Conversion of Seryl-tRNASec to
Selenocysteyl-tRNASec--
Ser-tRNASec was
converted to selenocysteyl-tRNASec in a reaction mixture
containing 100 mM Pipes (pH 6.7), 10 mM KCl, 10 mM magnesium acetate, 0.5 mM DTT, 5 mM ATP, 250 µM sodium selenite, 17 µM seryl-tRNASec, 0.1 mg/ml SelA, and 5.9 mg/ml SelD. After incubation at 37 °C for 35 min, 0.05 volume of 3 M sodium acetate (pH 4.6) and 0.05 volume of 200 mM DTT were added. The solution was thoroughly shaken with
1 volume of phenol equilibrated with 100 mM sodium acetate (pH 4.6) and centrifuged for 5 min at 16,000 × g. The
supernatant was then mixed with 0.1 volume of 3 M sodium
acetate (pH 4.6) and 1 volume of isopropyl alcohol and incubated for
1 h on ice. The precipitate was sedimented by centrifugation for
10 min at 16,000 × g, washed with 80% ethanol
containing 5 mM DTT, dried in vacuo, and
resolubilized in 10 mM sodium acetate (pH 4.6) containing 5 mM DTT. All steps were performed in a glove box under
anaerobic conditions. The concentration of
selenocysteyl-tRNASec was determined by performing parallel
reactions using sodium [75Se]selenite instead of the
nonradioactive compound and determining the radioactivity as described above.
Transient Kinetic Experiments--
Stopped-flow experiments were
performed in a High Tech Scientific instrument using an excitation
wavelength of 366 nm and an emission cutoff filter with an edge at 389 nm for monitoring the fluorescence from the methylanthraniloyl group.
For tryptophan fluorescence, an excitation wavelength of 289 nm and a
320-nm cutoff filter were used. For fluorescence resonance energy
transfer measurements, a combination of the 289-nm excitation
wavelength with the 389-nm filter were used. To monitor the
fluorescence of the fluorescein group, an excitation wavelength of 494 nm and a 530-nm cutoff filter were used. Since the protein stock
solutions were in 50% glycerol and relatively dilute, all solutions of
reactants were adjusted to 10% glycerol content. Experiments in which
the only source of glycerol was the protein stock solution, which resulted in a final glycerol concentration in the observation chamber
that was 
5%, led to very similar results for nucleotide association
kinetics, but with the disadvantage that there was a significant
disturbance of the optical signal at the beginning of the transient.
The buffer used contained 100 mM potassium phosphate (pH
7.0), 5 mM MgCl2, 2 mM DTT, 0.5 mM EDTA, and 10% glycerol. Primary data fitting was
performed with the software of the stopped-flow apparatus, and
secondary analysis was with GraFit Version 3.0 (Erithacus Software,
Staines, United Kingdom) and Scientist Version 2.01 (Micromath, Salt
Lake City, UT).
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RESULTS |
Interaction of mantdGTP and GTP with SelB--
Binding of GTP and
GDP derivatives with a methylanthraniloyl group in the ribose moiety to
SelB resulted in a significant increase (~130%) in the fluorescence
of the attached group. It was difficult to use this signal in
equilibrium titration experiments since titrating a fixed concentration
of the enzyme with increasing concentrations of fluorescent nucleotide
resulted in a strongly increasing background signal from excess unbound
nucleotide. The standard solution to this problem would be to titrate
an increasing concentration of protein to a fixed concentration of
fluorescent nucleotide. This was precluded by the low solubility of the
protein, which prevented preparation of a highly concentrated stock
solution. These problems did not prevent transient kinetic experiments
using the stopped-flow method, and an example of the fluorescence
transient seen on rapid mixing of SelB and mantdGTP is shown in Fig.
2A. mantdGTP was used for most
of the studies reported here since it was noticed that with mantGTP
(i.e. the ribose rather than the deoxyribose derivative),
biphasic association curves were obtained, and it was suspected that
this was due to the fact that mantGTP is a mixture of the 2'- and
3'-isomers (20). The monophasic transients observed with mantdGTP were
consistent with this interpretation.
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Fig. 2.
Kinetics of association of SelB with
mantdGTP. A, transient of the association of SelB (0.3 µM) with mantdGTP (19 µM) as monitored in
the stopped-flow apparatus. Fluorescence was detected by resonance
energy transfer from tryptophan to the mant group using 289 nm as the
excitation wavelength and detecting through a 389-nm cutoff filter. The
fitted line corresponds to an exponential function with a rate constant
of 3.29 s1. B, dependence of the
pseudo first-order rate constant obtained from experiments of the type
shown in A on the mantdGTP concentration. The fitted
straight line corresponds to values of 1.67 × 105
M1 s1
for k+1 and 0.11 s1
for k1 (as defined in Equation 1).
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Increasing the mantdGTP concentration while keeping the SelB
concentration constant led to a linear increase in the observed pseudo
first-order rate constant of association, as shown in Fig. 2B. In this type of experiment, the observed rate constant
is given by the following relationship (Equation 1),
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(Eq. 1)
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where k+1 and
k1 are the association and
dissociation rate constants, respectively in the interaction between the protein and the nucleotide. The slope of the fitted straight line
in Fig. 2B defines the value of k+1
as 1.67 × 105
M1 s1,
whereas the intercept on the y axis is small, but appears to be of the order of ~0.1 s1, giving an
approximate value for k1.
The value of k1 was determined
more exactly in a displacement experiment in which an excess of
unlabeled GTP was used to displace mantGTP. As shown in Fig.
3, the rate of mantdGTP release is indeed
relatively slow and has the value of 0.092 s1. Taking the two kinetic constants together
allows us to calculate a value of 0.55 µM for the
Kd value of mantdGTP. This is in reasonable
agreement with the value of 1.7 µM determined by
equilibrium dialysis using radioactively labeled GTP (6).
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Fig. 3.
Displacement of mantdGTP from SelB. A
complex of mantdGTP (4 µM) and SelB (1.2 µM) in one syringe of the stopped-flow apparatus was
mixed with GTP (200 µM) in the other. The fluorescence of
the mant group was excited at 366 nm and observed through a 389-nm
cutoff filter. Due to slight bleaching of fluorescence during the
measurement, a linear term was introduced into the single exponential
fit. The fitted line corresponds to a rate constant of 0.092 s1.
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Since it was of importance to determine the corresponding parameters
for the natural substrate GTP, intrinsic protein fluorescence was also
examined as a potential signal of nucleotide binding. A small
(~2.5%) increase in fluorescence occurred on GTP binding, and
although this was too small to be used conveniently for accurate equilibrium titrations, it could be used to monitor the association kinetics with SelB using the stopped-flow method. As can be seen from
the data of Fig. 4A, the
signal is noisy, but increasing the GTP concentration led to an obvious
increase in the rate of the transient seen. In Fig. 4B, it
is apparent that there is a linear dependence of the observed rate
constant on the GTP concentration, and the slope of the plot of the
rate constant against GTP concentration leads to a value of 2.16 × 105 M1
s1, which is similar to that of mantdGTP from
Fig. 2B. The rate constant for the association of unmodified
GTP could also be determined by using it as a competitor for mantdGTP
in experiments of the type described for Fig.
5. Here, the GTP concentration was
varied, and the mantdGTP concentration was held constant. The slope of the straight line fit to the data points defines the rate constant for
GTP association with SelB, which, in approximate agreement with the
results of Fig. 4B, has a value of 1.93 × 105 M1
s1.
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Fig. 4.
Kinetics of association of SelB with
GTP. A, association kinetics of SelB (0.6 µM) with GTP (16 µM) using tryptophan
fluorescence as a monitor (excitation wavelength of 289 nm; detection
through a 320-nm cutoff filter). The curve was fitted as a single
exponential, making allowance for significant bleaching of fluorescence
by including a linear term in the fit equation. The fitted line
corresponds to a rate constant of 3.85 s1.
B, dependence of the pseudo first-order rate constant
obtained from experiments of the type shown in A on the GTP
concentration. The fitted straight line corresponds to values of
2.16 × 105 M1
s1 for k+1 and 0.28 s1 for
k1.
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Fig. 5.
Competitive binding kinetics of SelB with
mantdGTP in presence of varying concentrations of GTP. The
concentrations of SelB and mantdGTP were 0.6 and 6 µM,
respectively. Fluorescence was detected as described in the legend Fig.
2. The fitted line corresponds to a value of 1.93 × 105 M1
s1 for k+1.
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The rate constant for GTP release was determined by displacement with
excess mantdGTP. Since this experiment could not be performed with a
very large excess of mantdGTP (because of the high background from
unbound mantdGTP), it was performed at two different concentrations of
mantdGTP, both in moderate excess over GTP, which was preincubated with
SelB. Since the measured rate constant in both cases was identical
within experimental error, we conclude that this represents the value
k1 for GTP (0.16 s1). Again, the calculated
Kd value (0.74 µM) is in good agreement with that determined previously by equilibrium dialysis (6).
Interaction of mantdGDP and GDP with SelB--
GDP and mantGDP
displayed quantitatively different kinetics of the interaction with
SelB when compared with GTP or mantGTP. Initial stopped-flow
experiments showed that the association reaction was faster than for
GTP or mantdGTP. Since the signal quality at the high concentrations of
mantGDP needed for these studies was not sufficient to detect the
deviation from monophasic behavior seen with mantGTP, the more readily
available mantGDP (and not mantdGDP) was used for these experiments.
Fig. 6A shows the dependence of the observed rate constant for association of mantGDP on the concentration. From the approximately linear dependence, values of
1.79 × 106 M1
s1 for the association rate constant and
14.25 s1 for the dissociation rate constant
were obtained. The rate constant for association of unmodified GDP was
obtained by examining the competing influence of GDP on the mantGDP
association, as described above for GTP, and the results are shown in
Fig. 6B. The value obtained from the slope of the fitted
straight line (1.12 × 106
M1 s1)
is similar to that for mantGDP and confirms that the association rate
constant is considerably greater than for GTP.
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Fig. 6.
Kinetics of association of SelB with mantGDP
and GDP. A, dependence of the pseudo first-order rate
constant for association of SelB (0.75 µM) with mantGDP
on the nucleotide concentration. Fluorescence was detected as described
in the legend to Fig. 2A. The slope of the fitted straight
line corresponds to a value of 1.79 × 106
M1 s1
for k+1, whereas the y axis intercept
gives a value of 14.25 s1 for
k1. B, competitive
binding kinetics of SelB (0.6 µM) with mantGDP (5 µM) in the presence of varying concentrations of GDP.
Detection was as described in the legend to Fig. 3. The fitted line
corresponds to a value of 1.12 × 106
M1 s1
for k+1.
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A further indication of the more rapid equilibration of GDP compared
with GTP was obtained from experiments in which GDP was used as a
competitor of mantdGTP binding in association reactions. In contrast to
the behavior shown in Fig. 5, where GTP competed with mantdGTP binding,
the first-order rate constant for mantdGTP binding decreased as the GDP
concentration was increased (data not shown). As discussed previously
(21), this type of behavior arises when the competing ligand is in
rapid equilibrium with its bound form on the time scale of the observed transient.
More accurate estimates of the dissociation rate constants for mantGDP
and GDP were obtained from displacement experiments, as shown in Fig.
7. Displacement of mantGDP by GDP could
be measured easily since a large excess of the nonfluorescent
unmodified nucleotide could be used. The value of the dissociation rate
constant obtained (13.9 s1) is consistent
with the magnitude of the intercept on the y axis in Fig.
6A (14.25 s1). GDP dissociation
was more difficult to measure due to the large background fluorescence
of a high concentration of mantGDP as displacing agent. The experiment
was repeated at two different concentrations of displacing agent, with
little effect on the measured rate constant, which was ~15
s1.
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Fig. 7.
Displacement of mantGDP from SelB. The
displacement of mantGDP from a complex generated by mixing 40 µM mantGDP with 1.2 µM SelB in one syringe
of the stopped-flow apparatus by GDP (1 mM) was monitored
by resonance energy transfer as described in the legend to Fig.
2A. The fitted line corresponds to a rate constant of 13.9 s1.
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The calculated Kd values for GDP and mantGDP are
13.4 and 7.8 µM, respectively. The results of the kinetic
experiments with GTP, GDP, and their fluorescent derivatives are
summarized in Table I.
Interaction of RNA Stem-Loops with SelB--
On interaction of
SelB with the 17-mer fdhF minihelix, there was a small
quenching of the intrinsic protein fluorescence. However, the size of
the signal change was too small to allow reliable kinetic or titration
experiments to be performed.
Detailed investigations of the interactions of the two RNA stem-loop
structures directing selenocysteine insertion in E. coli (Fig. 1) with SelB were possible using a fluorescently labeled construct. The fdhF minihelix bearing a fluorescein group at
the 5'-end displayed a significant increase in fluorescence intensity on interaction with the protein. The signal was stable enough to allow
a titration of SelB to a constant concentration of labeled fdhF minihelix RNA. This resulted in a maximal increase of
~7% in fluorescein fluorescence. The shape of the curve obtained
(Fig. 8A) already indicates a
high affinity for the interaction since at the concentration used (181 nM fluorescent fdhF minihelix), the almost
linear approach to the plateau reached at saturation suggests that the
Kd value must be much less than 181 nM.
Fitting the data to a quadratic equation leads to a
Kd value of 0.7 nM. However, this value
is not regarded as highly reliable since it will be determined by just
a few points as saturation is closely approached. The signal was not
stable enough to allow titrations at significantly lower concentrations
of RNA. However, the kinetic studies described below confirm that the
Kd value is in the range of 1 nM.
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Fig. 8.
Interaction of SelB with SECIS elements.
A, titration of SelB to fluorescein-labeled fdhF
minihelix RNA (181 nM). The measurements were performed
with an SLM8000 spectrophotometer (Aminco, Silver Spring, MD).
Excitation was at 490 nm, and emission was detected at 520 nm. The
fitted line corresponds to a Kd value of 0.7 nM. B, displacement of the fluorescein-labeled
fdhF minihelix (181 nM) from a complex with SelB
(198 nM) by the unlabeled fdhF minihelix ( )
or fdnG stem-loop ( ). Fluorescence was detected as
described for A. The fitted line corresponds to a
Kd value of 1.26 nM for the labeled
stem-loop construct (from the kinetic data of Table II) and a fitted
Kd value of 1.36 nM for the unlabeled
fdhF minihelix. The fitted Kd value for
the fdnG stem-loop (fitted line not shown) is 1.38 nM.
|
|
To test whether the fluorescent label on the RNA has an influence on
the interaction with SelB, a competitive titration was performed in
which fluorescent fdhF minihelix was displaced from its
preformed complex with SelB by a large excess of unlabeled fdhF minihelix. As shown in Fig. 8B, the labeled
RNA could be completely displaced. This provides evidence that the
binding is purely competitive and excludes the possibility of
nonspecific binding of the labeled RNA. Fitting the data using the
program Scientist led to the conclusion that the affinity of SelB for the fdhF minihelix is almost unaffected by the modification.
Using a Kd value of 1.26 nM for
fluorescent fdhF minihelix, the Kd for
the unmodified minihelix was calculated to be 1.36 nM. Fig.
8B also shows that displacement by the other stem-loop
construct examined (fdnG) led to identical results, and the
derived Kd value was 1.38 nM. We thus
conclude that the interaction of the stem-loop structures with SelB
occurs with nanomolar affinity and is unaffected by a large fluorescent label at the 5'-end of the structure.
In further experiments, the fluorescently labeled RNA was used to
investigate the kinetics of the interaction with SelB. Fig. 9A shows the results of
monitoring the association reaction in the stopped-flow apparatus. The
rate constant for the single exponential transient observed showed a
linear dependence on the concentration of SelB at a constant
fluorescent fdhF minihelix concentration (Fig.
9B). The linear dependence allows calculation of the
second-order rate constant for the interaction, which is rapid and
approaches the diffusion-controlled limit (Table
II). The fact that the fit to the points
obtained passes through the origin of the graph within the limits of
experimental error shows that the dissociation rate is very low. It
could be measured directly by displacement of fluorescent
fdhF minihelix from its complex by an excess of unlabeled
fdhF minihelix (Fig.
10A). Using the value for
the dissociation rate constant obtained (0.28 s1) together with the association rate
constant (Table II), a Kd value of 1.26 nM can be calculated for the interaction, confirming the
conclusion arising from the titration experiments of Fig. 8A
that the Kd value is of the order of 1 nM.
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Fig. 9.
Kinetics of association of SelB with
fluorescein-labeled fdhF minihelix. A,
association transient of SelB (0.125 µM) and the
fluorescein-labeled fdhF minihelix (45.3 nM).
The fluorescence of the fluorescein group was excited at 494 nm and
detected through a 530-nm cutoff filter. The fitted line corresponds to
a monoexponential function with a rate constant of 29.6 s1. B, dependence of the pseudo
first-order rate constant for the association of SelB with the labeled
fdhF minihelix on the SelB concentration. The fitted
straight line corresponds to values of 2.23 × 108 for
k+1 and 0.75 for
k1.
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|
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Fig. 10.
Displacement of fluorescein-labeled
fdhF minihelix from SelB and SelB-(472-614).
A, displacement of the fluorescein-labeled fdhF
minihelix from SelB by the unlabeled fdhF minihelix. A
complex of SelB (0.25 µM) and the labeled fdhF
minihelix (0.14 µM) in one syringe was mixed with 15 µM unlabeled fdhF minihelix in the other
syringe (curve a). Fluorescence was detected as described in
the legend to Fig. 9A. The experiment represented by
curve b was performed under the same conditions, but 4 µM GTP and 2.5 µM
selenocysteyl-tRNASec were included in both syringes, and
the labeled fdhF minihelix concentration was 0.21 µM. Fitting of the data to a monoexponential function
defined the rate constants for the reactions as 0.28 s1 (curve a) and 0.043 s1 (curve b). B,
displacement of the fluorescein-labeled fdhF minihelix from
SelB-(472-614) by the unlabeled fdhF minihelix. A complex
of SelB-(472-614) (0.31 µM) and the labeled
fdhF minihelix (0.14 µM) in one syringe was
mixed with 15 µM unlabeled fdhF minihelix in
the other syringe. Fluorescence was detected as described in the legend
to Fig. 9A. The fitted line corresponds to an exponential
function with a rate constant of 0.037 s1.
|
|
It was possible to obtain an approximate value for the association rate
constant for unmodified RNA by examining the effect of the unlabeled
fdhF stem-loop on the association kinetics of the labeled
molecule. As shown in Fig. 11,
inclusion of unlabeled stem-loop RNA with the fluorescently labeled
fdhF minihelix resulted in a measurable effect on the
association kinetics. A global analysis of the curves shown Fig. 11
leads to a value of ~108
M1 s1
for the association rate constant for the unlabeled construct.
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Fig. 11.
Kinetics of association of
fluorescein-labeled fdhF minihelix with SelB in the
presence of different concentrations of unlabeled fdhF
minihelix. The concentrations of fluorescein-labeled
fdhF minihelix (0.136 µM) and SelB (0.128 µM) were constant, whereas the concentration of unlabeled
minihelix was varied as indicated in the graph. Fluorescence was
detected as described in the legend to Fig. 9A. The fitted
lines represent association transients calculated by global analysis of
the curves using the program Scientist. They define the values of the
rate constants for the association and dissociation reactions as
k+1 = 2.39 × 108
M1 s1
and k1 = 0.28 s1 for the labeled fdhF minihelix
and k+1 = 1.09 × 108
M1 s1
and k1 = 0.28 s1 for the unlabeled fdhF
minihelix.
|
|
To examine possible interaction or communication between the nucleotide
and stem-loop binding sites, the experiments of Fig. 9 were repeated in
the presence of GDP and GTP. As can be seen from the values for the
association and dissociation rate constants given in Table II, there
was no detectable influence of the state of the nucleotide binding site
of SelB on the kinetics of the interaction.
In complementary experiments, the effect of stem-loop RNA on the
interaction of the protein with nucleotides was examined. As shown in
Table III, the values for the association
and dissociation rate constants for mantdGTP and mantGDP obtained in
the presence of the fdnG stem-loop at a concentration that
is enough to saturate the mRNA binding site are indistinguishable
from those obtained in the absence of RNA.
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Table III
Rate constants for the interaction of guanosine nucleotides with SelB
in the presence of the fdnG stem-loop structure
|
|
Interaction of the RNA Binding Domain (Domain IVb) with Stem-Loop
RNA--
To determine to what extent the RNA binding properties of
this domain are affected by the rest of the protein, we examined the
interaction of the separately expressed domain with the stem-loop structure. Using the fluorescein-labeled fdhF minihelix and
the isolated domain in the stopped-flow apparatus, the association kinetics were nearly identical to those seen with full-length SelB
(ka = 2.58 × 108
M1 s1)
(data not shown). However, the dissociation kinetics were slowed significantly. The rate constant for dissociation was 0.037 s1 (Fig. 10B), in comparison with
0.28 s1 for the dissociation from full-length
SelB. Thus, the affinity of the isolated fragment
(Kd = 0.14 nM) is increased in
comparison with full-length SelB (Kd = 1.26 nM).
An indication that this difference in the properties of SelB and domain
IVb might be of mechanistic importance comes from the second curve
shown in Fig. 10A. We have not yet been able to identify a
useable direct signal to monitor the interaction of selenocysteyl-tRNASec with SelB, but a clear indication of
its binding arises from the observation that in the presence of the
charged tRNA, the rate of dissociation of the fdhF minihelix
is slowed significantly. In quantitative terms, it is slowed to a rate
that is nearly indistinguishable from that of the stem-loop construct
from isolated domain IVb, suggesting that whatever the nature of the
interactions of domain IVb with the other domains of SelB that lead to
a reduction of its affinity for the stem-loop structure, these
interactions are removed or relieved when
selenocysteyl-tRNASec is bound.
| |
DISCUSSION |
The results presented show that SelB displays a relatively low
affinity for GTP and GDP in binary complexes between the protein and
the nucleotides. In contrast to EF-Tu, the GTP affinity is greater (by
an order of magnitude) than the GDP affinity. This difference is mainly
due to a dramatic difference in the GDP affinities in the two systems,
with the GTP affinities being more similar. Although GDP is bound more
weakly than GTP, its kinetics of interaction with SelB are considerably
faster. Thus, the association rate constant is almost an order of
magnitude faster for GDP than for GTP (cf. a factor of 3 for
EF-Tu) (22). In terms of affinity, this effect is more than compensated
for by the fact that GDP is released ~2 orders of magnitude faster
than GTP so that GTP binds more strongly than GDP.
The weaker binding of GDP compared with GTP and, in particular, the
high dissociation rate constant are consistent with the idea that an
exchange factor for SelB is not required since the intrinsic GDP
dissociation rate is of the same order of magnitude as the exchange
factor-catalyzed dissociation rate with EF-Tu. Somewhat similar
properties have been seen for the E. coli signal recognition
particle receptor FtsY (23), i.e. weak affinities for GTP
and GDP and high exchange rates. However, in this case, GDP is bound
more strongly than GTP and exchanges rapidly in comparison with EF-Tu
or the Ras family of proteins, but more slowly than GTP.
When the kinetic results obtained in this work are compared with those
for other GTPases, the most striking difference observed is the rapid
rate of spontaneous GDP dissociation. With the exception of FtsY, all
other GTPases involved in signal transduction and regulation that have
been characterized in detail with respect to their kinetic properties
have slow (~103
s1 for EF-Tu) (22) or very slow
(106 to 104
s1 for Ras and Ras-related proteins) (21, 24)
GDP dissociation rates, which are accelerated dramatically by exchange
factors. The same is also true for the heterotrimeric G-proteins; and
in the case of GTPases involved in signal transduction, it is easy to
understand why GDP release must occur in a manner that is dependent on
activation or recruitment of an exchange factor, this process being
dependent, in general, on primary activation events in the signal
transduction pathway. For ribosomal elongation factors, the requirement
for an exchange factor is more difficult to rationalize; and in the
case described here, SelB appears to function without one, proving that
the kinetic properties of the GTPase can be designed so that the
principle already known from other elongation factors can work even if
the GDP produced concomitantly with peptide bond formation is bound
weakly to the factor and dissociates spontaneously, allowing rebinding
of GTP and the start of another cycle.
The experiments reported on the kinetics of the interaction of
stem-loop constructs with SelB show that this is a high affinity interaction, with a Kd value of ~1.3
nM. The association rate constant approaches the
diffusion-controlled limit, and dissociation in the absence of tRNA
occurs with a half-life of a few seconds. Neither the association nor
the dissociation kinetics are affected by the presence or the nature of
the guanosine nucleotide at the active site of the GTPase domain.
Conversely, as is to be expected based on thermodynamic considerations,
the presence of a stem-loop construct at its binding site does not
affect the kinetics of the nucleotide interaction with SelB. Thus,
there is no coupling between the nucleotide binding site and the RNA
binding site, meaning that recruitment of SelB to its binding site on
mRNA is not dependent on the state of the nucleotide binding site.
Although we were not able to identify a usable spectroscopic signal for
the interaction of selenocysteyl-tRNASec with SelB, there
was a clear indication that the rate of dissociation from the stem-loop
structure was slower when the charged tRNA was bound, suggesting an
interaction between the two binding sites. The fact that a similar
slowing down of the dissociation rate from mRNA was seen with the
isolated domain IVb suggests that an interaction between the tRNA
binding domain and the mRNA binding domain occurs in the absence of
tRNA that is weakened or removed when tRNA is bound. Further studies
with additional signals will be needed to clarify the nature of the
interactions between the two different types of RNA binding sites. The
possible functional significance of this effect is that the full
affinity of SelB for its binding site on RNA is only realized when
selenocysteyl-tRNASec is bound. Binding of the charged tRNA
thus increases the stability of the
SelB·GTP·selenocysteyl-tRNASec·mRNA quaternary
complex and thereby favors the conformation of SelB able to interact
with the ribosome. This interaction triggers GTP hydrolysis and most
probably the release of the tRNA in the vicinity of the ribosomal A
site (4). As a consequence, there will be an increase in the rate of
dissociation of SelB from its mRNA binding site. This is required
for the translation of downstream codons since SelB complexed to its
SECIS site exerts a considerable kinetic barrier to the processivity of
translation (25).
| |
ACKNOWLEDGEMENT |
We thank Andrea Beste for the synthesis of
mant nucleotides.
| |
FOOTNOTES |
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to
A. B.) and by the Max Planck Society.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
49-231-1206381; Fax: 49-231-1206229; E-mail:
goody@mpi-do.mpg.de.
Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M002496200
| |
ABBREVIATIONS |
The abbreviations used are:
Sec, selenocysteine;
DTT, dithiothreitol;
Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid);
mantdGT(D)P, 3'-methylanthraniloyl-2'-deoxy-GT(D)P;
mantGT(D)P, 2'(3')-methylanthraniloyl-GT(D)P;
EF-Tu, elongation factor Tu;
SECIS, selenocysteine insertion sequence.
| |
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