From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication, December 18, 2002, and in revised form, March 10, 2003
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ABSTRACT |
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Initiator tRNAs are used exclusively for
initiation of protein synthesis and not for elongation. We show that
both Escherichia coli and eukaryotic initiator tRNAs have
negative determinants, at the same positions, that block their activity
in elongation. The primary negative determinant in E. coli
initiator tRNA is the C1xA72 mismatch at the end of the acceptor stem.
The primary negative determinant in eukaryotic initiator tRNAs is
located in the T A special methionine tRNA is used for the initiation of protein
synthesis in all organisms studied (1-3). The initiator tRNA is used
for the initiation of protein synthesis, whereas the elongator tRNA is
used for the insertion of methionine into internal peptidic linkages.
Because of their unique function, initiator tRNAs, tRNAfMet
in eubacteria and tRNA Initiator tRNAs also possess unique sequence and/or structural features
that are absent in elongator tRNAs. One of the unique features of
eubacterial initiator tRNAfMet (Fig. 1) is the presence of
a base pair mismatch between nucleotides 1 and 72 in the acceptor stem
(C1xA72). Elongator tRNAs have Watson-Crick base pairs between these
positions. It was shown earlier (10, 11) that this mismatch in the
acceptor stem is a primary determinant for exclusion of
tRNAfMet from elongation. U1 and G72 mutations that
generate a Watson-Crick base pair at this position allow the mutant
tRNAs to bind to EF-Tu and act as elongators, whereas a U1G72 double
mutation, which generates a U·G wobble base pair, does not (11,
12).
Eukaryotic initiator tRNAs also have highly conserved nucleotides at
position 1 and 72. In contrast to the C1xA72 mismatch in the bacterial
initiator tRNA, eukaryotic initiator tRNAs have a A1:U72 base pair.
In vivo and in vitro work on eukaryotic initiator tRNAs has shown that the primary determinant for preventing activity of
these tRNAs in elongation resides in the sequence/structure of the
T Another tRNA that does not bind to E. coli EF-Tu is the
selenocysteinyl-tRNASec, which binds to its own elongation
factor encoded by the selB gene (23). In this tRNA
also, the primary determinant, which prevents the tRNA from binding to
EF-Tu, has been located in the T Although the U1 and G72 mutants of E. coli
tRNAfMet are active in elongation, they are less active
than the E. coli elongator methionine tRNA
(tRNAMet). This finding raised the question of whether
there are other determinants in the E. coli
tRNAfMet which further limit its activity in elongation. In
particular, in view of the results above with eukaryotic initiator
tRNAs and tRNASec, we have investigated whether the
putative secondary determinant is located in the T This paper describes studies on the role of the U50·G64 wobble base
pair in the T Mutant tRNA Genes--
The mutant tRNAs used in this work were
all derived from tRNA Purification of Enzymes and Proteins--
E. coli
strain containing the pET24C(+) plasmid with the EF-Tu gene (27) was
kindly provided by Dr. Linda Spremulli (University of North Carolina,
Chapel Hill). Plasmid DNA was isolated from this strain and
reintroduced into E. coli BL21DE3 strain for expression of
His6-tagged EF-Tu. EF-Tu was purified from a 1-liter
culture of the BL21DE3 transformants grown in LB containing kanamycin (10 µg/ml) and induced for 3 h by the addition of 1 mM isopropyl-
Recombinant His6-tagged glutaminyl-tRNA synthetase (GlnRS)
was overproduced in E. coli M15 carrying the pQE60-QRS
vector and purified from a 1-liter culture grown in LB containing 50 µg/ml carbenicillin using a Cobalt Talon column (5-ml bed volume), as described for EF-Tu except that the GlnRS lysis buffer contained 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 8 mM Regeneration of EF-Tu with GTP--
EF-Tu·GTP was regenerated
from EF-Tu·GDP by incubation at 37 °C for 3 h in a solution
containing 50 mM Tris-HCl, pH 7.4, 150 mM
NH4Cl, 10 mM MgCl2, 3 mM phosphoenolpyruvate, 40 international units of pyruvate
kinase, 200 µM GTP, and 5 mM dithiothreitol. EF-Tu·GDP concentration in the regeneration mixture was 2-4
µM. Regenerated EF-Tu·GTP was chilled on ice for 15 min
and immediately used for KD or
koff assay with aminoacyl-tRNAs labeled with
[3H]Gln or [35S]Met.
Purification of Wild Type and Mutant
tRNAfMet--
E. coli B105 transformants
carrying the mutant tRNAfMet genes were grown in 1 liter of
2YT medium containing 50 µg/ml ampicillin. Cells were suspended in a
solution containing 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM EDTA at 1 g of
wet pellet per 8 ml of buffer. The suspension was mixed vigorously for
10 min with an equal volume of water-saturated phenol, and the phases
were separated by centrifugation. Nucleic acids in the aqueous layer were precipitated by addition of sodium acetate, pH 5.0, to 0.1 M followed by 2.5 volumes of 95% ethanol and storage for
2 h at Aminoacylation of tRNAs with [3H]Gln and
[35S]Met--
The reaction mixture (100 µl) contained
150 mM NH4Cl, 10 mM
MgCl2, 10 µg/µl bovine serum albumin, 0.1 mM EDTA, 20 mM imidazole, pH 7.6, 2.5 mM ATP, 2.0 A260 units of
gel-purified tRNAs (wild type, G72, C50/G72, and A64/G72 mutant
tRNAfMet), 50 µM [35S]Met
(specific activity 12 mCi/µmol), and 5 µg of MetRS. Aminoacylation of the U35A36, U35A36/G72G73, U35A36/C50/G72G73, and the
U35A36/A64/G72G73 mutant tRNAs with [3H]glutamine was
carried out similarly except that the reaction volume was 300 µl and
contained 5 mM dithiothreitol, 200 µM
[3H]glutamine (specific activity, 3.35 mCi/µmol,
~7,370 dpm/pmol), and 55 µg of GlnRS instead of
[35S]methionine and MetRS. In both cases, aminoacylation
was carried out at 37 °C for 15 min, and the reaction was stopped by
cooling to 0 °C, adding 0.1 M sodium acetate, pH 5.0, and extracting with phenol equilibrated to pH 5.0 with sodium acetate.
The aqueous layer was then extracted with phenol/chloroform (1:1, v/v)
and finally chloroform, and the aminoacyl-tRNA in the aqueous layer was
precipitated with ethanol and recovered by centrifugation. The pellet
was washed twice with 70% ethanol at 4 °C, air-dried, and dissolved
in 100 µl of ice-cold 5 mM sodium acetate, pH 5.0. The
solution was centrifuged at 4 °C through Sephadex G-25 spin columns
pre-equilibrated with 5 mM sodium acetate, pH 5.0, to separate aminoacyl-tRNA from ATP and free amino acids. Analysis of the extent of aminoacylation of tRNAs in vivo was carried
out as described previously (26).
Assay for Suppression in Vivo--
E. coli
CA274 transformants expressing the various mutant initiator tRNA
genes were grown at 37 °C in 5 ml of LB medium containing 50 µg/ml
ampicillin, until the A600 reached 0.8-1.0. At
this point, cultures were induced by adding 1 mM
isopropyl- Assay for Immunoblot Analysis of Bacterial Extracts with
Anti- Measurement of KD for the EF-Tu·GTP·Aminoacyl-tRNA
Ternary Complex--
We used a modification of the procedure described
by LaRiviere et al. (31). Sequentially diluted samples of
reconstituted EF-Tu·GTP (diluted from 1-2 µM to
1.95-3.9 nM in steps of 2-fold in buffer K: 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2,
150 mM NH4Cl, 100 µM GTP, 0.5 mM phosphoenolpyruvate, 10 mM dithiothreitol, and 40 µg/ml pyruvate kinase) were mixed with an equal volume (25 µl) of 3H- or 35S-labeled aminoacyl-tRNA
(20-40 nM), and the mixture (50 µl) was incubated for 20 min on ice to allow equilibration between bound and free forms of
aminoacyl-tRNA and EF-Tu·GTP. For each aminoacyl-tRNA, 12 such
samples including no EF-Tu·GTP control were simultaneously treated
with 5 µl of RNase A solution (Sigma, 0.2 mg/ml) for 30 s on
ice. RNase treatment was stopped by addition of 5 µl of a 1 mg/ml
solution of total E. coli tRNA (Sigma) followed by 25 µl
of cold 20% trichloroacetic acid. The samples were filtered under
vacuum through a sheet of Millipore membrane (HAWP, 0.45 µm) mounted
on a Bio-DotTM apparatus (Bio-Rad). The membrane was washed
3 times with 200 µl each of 5% trichloroacetic acid, removed from
the Bio-DotTM apparatus, immersed briefly (5 min) in
ice-cold 95% ethanol, and air-dried. The membrane fragments
corresponding to individual samples were cut out and placed into
scintillation vials for counting of [35S]Met or
[3H]Gln radioactivity.
Measurements of Off Rates of the EF-Tu·GTP·Aminoacyl-tRNA
Ternary Complexes--
The incubation mixture (100 µl) contained 4 µM EF-Tu·GTP and 40 nM aminoacyl-tRNA
(3H- or 35S-labeled) in buffer K. The mixture
was incubated on ice for 20 min and then treated with 10 µl of RNase
A (0.4 mg/ml, Sigma). Aliquots (10 µl) were taken out every 10 min
(from 0 to 90 min) and immediately transferred to the wells, on a
96-well assay plate, containing 100 µl of pre-chilled 10%
trichloroacetic acid with 2% casamino acids. The precipitated
radioactivity was collected by filtration under vacuum as described
above and counted.
Development of Experimental Data--
For calculation of
KD, EF-Tu concentrations were plotted as
x axis (in nM), and the amounts of ternary
complex (in nM) (calculated from counts/min of
[3H]Gln or [35S]Met in corresponding
samples), as y axis. KD values were
calculated with the help of Kaleidagraph application program (Synergy
software), from the curve fit based on Equation 1,
Mutant tRNAs Used--
Fig. 1 shows
the mutant tRNAs used in this work. To examine the possible role of
U50·G64 wobble base pair as a secondary determinant for restricting
the activity of the E. coli initiator tRNA in elongation,
this base pair was mutated to either a C50:G64 or U50:A64 base pair,
and these mutations were combined with mutations in the acceptor stem
and the anticodon sequence. The G72 mutation in the acceptor stem
allows the E. coli initiator tRNA to act as an elongator
(10), and the U35A36 mutation in the anticodon sequence allows the tRNA
to read the amber termination codon UAG. As a result the U35A36/G72
mutant tRNA is an amber suppressor in E. coli. Because of
the anticodon sequence change, the U35A36 and U35A36/G72 mutant tRNAs
are now aminoacylated with glutamine instead of methionine (32, 33).
However, the G72 mutation in the acceptor stem makes the U35A36/G72
mutant tRNA a poorer substrate for the E. coli
glutaminyl-tRNA synthetase (GlnRS) than the U35A36 mutant tRNA (11,
34). Therefore, for some experiments, the G72 mutation was also
combined with G73 mutation to make the mutant tRNAs better substrates
for the E. coli GlnRS.
Activity of the Mutant tRNAs in Elongation in E. coli--
The
activity of the mutant tRNAs in elongation in vivo was
measured by their activities in suppression of an amber mutation in the
Immunoblot analyses on cell extracts confirm the above results and show
that the increased activity of
It is extremely unlikely that the increased activity of the mutant
tRNAs carrying the C50 and A64 changes is due to an effect on
formylation of the tRNAs. First, biochemical (35) and structural studies (36) have shown that interactions of methionyl-tRNA formyltransferase (MTF) with the initiator tRNA are restricted to the
acceptor stem and the D stem. Second, the U35A36/G72 and the
U35A36/G72G73 mutant tRNAs are extremely poor substrates for MTF, and
there is no detectable formylation of these tRNAs in E. coli
(30, 37).
Extent of Aminoacylation of Mutant tRNAs in E. coli--
One
possible explanation of the increased activity in elongation of tRNAs
carrying the C50 or A64 mutations is increased aminoacylation of these
mutant tRNAs by GlnRS. To investigate this possibility, total tRNA
isolated under acidic conditions from E. coli CA274 transformants expressing the various mutant tRNAs was separated on an
acid-urea polyacrylamide gel, and the uncharged and aminoacylated forms
of the mutant tRNAs were detected by RNA blot hybridization using a
deoxyribo-oligonucleotide probe complementary to sequences in the
anticodon stem and loop common to all of the mutant tRNAs (Fig.
3). A probe for the endogenous tyrosine
tRNA (tRNATyr) was used as an internal control. The amounts
of uncharged and aminoacylated forms of the mutant tRNAs (bands
A and B of Fig. 3) were quantified using a
PhosphorImager, and the pixels were used to calculate the percent of
each mutant tRNA that is aminoacylated in the cell. The amount of the
aminoacylated form of each of the mutant tRNAs was then normalized to
the amount of total tRNATyr present in each sample
(bands C and D of Fig. 3). It can be seen (Table
II) that within one set of mutants, the
U35A36/G72 or the U35A36/G72G73, introduction of C50 or A64 mutations
had little effect on the extent of aminoacylation of the tRNA and on
the relative content of the mutant aminoacyl-tRNAs in the cell. As expected, the extent of aminoacylation of tRNAs carrying the
U35A36/G72G73 mutation is higher than that of tRNAs carrying the
U35A36/G72 mutation. These results suggest that the increased activity
in elongation of the tRNAs carrying the C50 or the A64 mutations is due
to their increased activity at a step following aminoacylation of the
tRNA. This could be due either to an increased affinity of the mutant
aminoacyl-tRNAs for EF-Tu or for the ribosomal A site.
Binding Affinity of EF-Tu·GTP for the Mutant
Aminoacyl-tRNAfMet--
Two sets of mutant tRNAs were used
for this work. One set contains the G72 and G73 mutations in the
acceptor stem and the U35A36 mutation in the anticodon sequence (Fig.
1A). These mutant tRNAs were aminoacylated in
vitro with [3H]glutamine using E. coli
GlnRS. For maximal aminoacylation of the mutant tRNAs with E. coli GlnRS, the tRNAs with the G72G73 mutations were used rather
than the ones with the G72 mutation alone (11). The other set of mutant
tRNAs used contained just the G72 mutation in the acceptor stem (Fig.
1B). These tRNAs were aminoacylated in vitro with
[35S]methionine using E. coli MetRS. The
G72G73 mutant tRNA could not be used because this tRNA is not
aminoacylated by E. coli MetRS (38).
A ribonuclease protection assay was used to measure
K Stability of EF-Tu·GTP·Aminoacyl-tRNA Ternary
Complexes--
In an extensive analysis of the binding of several
aminoacyl-tRNAs to EF-Tu·GTP, Uhlenbeck and co-workers (31) have
noted a strong correlation between the dissociation rate constants
(koff) of the EF-Tu·GTP·aminoacyl-tRNA
ternary complexes and the KD values. This suggests
that kon, the association rate constant, is the
same for many of the aminoacyl-tRNAs. Therefore, the
koff values of the ternary complexes could be
equally useful parameters in comparing the affinity of EF-Tu·GTP for
a series of related mutant aminoacyl-tRNAs. Furthermore, although the
measurement of KD values is sensitive to errors in
estimate of the amount of active EF-Tu·GTP in a preparation and the
fraction of aminoacyl-tRNA that is active in binding to EF-Tu,
measurement of koff does not suffer from the
limitations. Additionally, measurement of koff
values allows one to determine whether the extremely poor binding of
wild type E. coli Met-tRNAfMet to EF-Tu·GTP
(Fig. 4B) is due to a very low association rate constant of
binding or due to extreme instability of the ternary complex. We have,
therefore, measured the koff of EF-Tu·GTP
complexes with the various mutant [3H]Gln- and
[35S]methionyl-tRNAs to obtain an independent and
possibly a more accurate estimate of the binding affinities of the
various aminoacyl-tRNAs toward EF-Tu·GTP.
Wild type and mutant aminoacyl-tRNAs (40 nM) were mixed
with an excess of E. coli EF-Tu·GTP at a high
concentration (4 µM) such that essentially all of the
aminoacyl-tRNAs were in the form of a ternary complex. RNase A was
added to the incubation mixture, and aliquots were taken every 10 min
(except for Met-tRNAfMet in which case it was every 5 s) for the measurement of residual trichloroacetic acid-precipitable
radioactivity. Dissociation of the ternary complex renders the
aminoacyl-tRNAs susceptible to RNase A cleavage. Therefore, the
time-dependent loss of trichloroacetic acid-precipitable
radioactivity can be used to measure koff, the dissociation rate constant of the ternary complex (see
"Experimental Procedures").
Fig. 5, A and B,
shows representative examples of the dissociation rates of the
amino acyl~tRNA·EF-Tu·GTP ternary complexes for the two
sets of mutant tRNAs. Table IV lists the
mean koff values. With the U35A36 mutant tRNA
that is aminoacylated with glutamine, introduction of the G72G73
mutation increases the stability of the ternary complex by about
3.5-fold. Introduction of additional mutations in the U50·G64 wobble
base pair leads to further stabilization of the complex by an
additional factor of 2.4-2.6-fold. With the wild type initiator tRNA
that is aminoacylated with methionine, and that binds extremely poorly
to EF-Tu·GTP, introduction of the G72 mutation leads to a large
increase in stability of the ternary complex, by a factor of
~45-fold. Introduction of additional mutations in the U50·G64
wobble base pair increases further the stability of the ternary
complex, although less than that for the corresponding mutant tRNAs
that are aminoacylated with glutamine. As with the
K
The very rapid hydrolysis of wild type Met-tRNAfMet by
RNase A (Fig. 5B) led us to investigate whether this
aminoacyl-tRNA forms a ternary complex at all with EF-Tu·GTP or
whether the time-dependent hydrolysis simply reflects the
rate of RNase A cleavage of unbound Met-tRNAfMet. To
distinguish among these possibilities, Met-tRNAfMet was
treated with RNase A in the presence or absence of EF-Tu·GTP. Results
in Fig. 6 show that EF-Tu·GTP protects
the Met-tRNAfMet from RNase A cleavage. In the absence of
EF-Tu·GTP, RNase A cleavage of Met-tRNAfMet is extremely
fast. Thus, the Met-tRNAfMet does form a complex with
EF-Tu·GTP; however, the complex is extremely unstable with a
half-life of ~15 s. This extreme instability of the
EF-Tu·GTP·Met-tRNAfMet ternary complex explains our
inability to detect the formation of such a complex in the equilibrium
binding curves used to measure KD values (Fig.
4B). The 30-s treatment with RNase A used to hydrolyze all
of the unbound Met-tRNAfMet would have hydrolyzed more than
75% of the Met-tRNA in the extremely unstable ternary complex.
The primary negative determinant blocking activity of E. coli initiator tRNAfMet in elongation is the C1xA72
mismatch at the end of the acceptor stem. This is due to perturbation
of the RNA helical structure caused by the C1xA72 mismatch (10). Here
we have shown that the E. coli initiator tRNA has a
secondary negative determinant, the 50·64 wobble
base pair, that is located in the TC stem, whereas a secondary negative determinant is
the A1:U72 base pair at the end of the acceptor stem. Here we show that
E. coli initiator tRNA also has a secondary negative
determinant for elongation and that it is the U50·G64 wobble base
pair, located at the same position in the T
C stem as the primary
negative determinant in eukaryotic initiator tRNAs. Mutation of the
U50·G64 wobble base pair to C50:G64 or U50:A64 base pairs increases
the in vivo amber suppressor activity of initiator tRNA
mutants that have changes in the acceptor stem and in the anticodon
sequence necessary for amber suppressor activity. Binding assays of the
mutant aminoacyl-tRNAs carrying the C50 and A64 changes to the
elongation factor EF-Tu·GTP show marginally higher affinity of the
C50 and A64 mutant tRNAs and increased stability of the
EF-Tu·GTP· aminoacyl-tRNA ternary complexes. Other results show a
large effect of the amino acid attached to a tRNA, glutamine
versus methionine, on the binding affinity toward
EF-Tu·GTP and on the stability of the EF-Tu·GTP·aminoacyl-tRNA ternary complex.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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C stem, with the A1:U72 base pair acting as a secondary determinant
(13-17). For example, fungal and plant initiator tRNAs have a bulky
2'-O-phosphoribosyl modification on the ribose of nucleotide
64 in the T
C stem (18-20). Removal of this bulky modification allows the initiator tRNA to bind to eEF1 and to act as an elongator (13-15). Vertebrate initiator tRNAs lack this bulky modification (21,
22). However, mutations of base pairs 50:64 and 51:63 allow the mutant
tRNA to act as an elongator tRNA in vivo in mammalian cells
suggesting that in the vertebrate initiator tRNAs it is the
sequence-dependent perturbation of the T
C stem that
blocks the tRNA from acting as an elongator (17). Coupling of the 50:64 and 51:63 base pair mutations with A1:U72
G1:C72 mutation further increased the activity of the tRNA in elongation, suggesting that the
rather weak A1:U72 base pair in the acceptor stem was a secondary determinant.
C stem (24).
C stem of the tRNA.
C stem of E. coli tRNAfMet. A
wobble base pair at this site or in adjacent sites in the T
C stem is
found in most bacterial, mitochondrial, and chloroplast initiator tRNAs
(25). (An update of this compilation can be found in
www.uni-bayreuth.de/departments/biochemie/trna//.) We have
mutated the U50·G64 wobble base pair to C50:G64 (C50 mutant) or to
U50:A64 (A64 mutant) Watson-Crick base pairs and combined this to (i)
mutations (U1, data not shown; G72 or G72G73) in the acceptor stem,
which allow the tRNA to act in elongation; and (ii) mutations (U35A36)
in the anticodon sequence, which allow the tRNAs to read the amber
(UAG) termination codon. We show that mutations of the 50·64
wobble base pair to either C:G or U:A base pairs increases the activity
in elongation of all of the mutant tRNAs in vivo. In
addition, we have investigated whether this increase in elongation
activity is due to increased affinity toward EF-Tu.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
C stem of tRNA (Fig. 1) were introduced by
QuickChange site-directed mutagenesis with the appropriate mutagenic
primers, using Pfu DNA polymerase, according to Stratagene.
-thiogalactoside. Wet bacterial pellet was
suspended in EF-Tu lysis buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10 mM MgCl2, 7 mM
-mercaptoethanol, 50 µM GDP, 0.5 mM phenylmethylsulfonyl fluoride) at 1 g/15 ml, and the
bacteria were lysed by freeze-thawing in the presence of 0.1 mg/ml of
lysozyme. The lysate was treated with DNase I and clarified by
centrifugation (12,000 × g) for 30 min at 4 °C. The
supernatant was mixed with 2 ml of Talon-Sepharose CO2+
metal affinity resin (Clontech) and nutated for 30 min at 4 °C. The resin was allowed to settle and washed three times
with 5 volumes of EF-Tu lysis buffer; the slurry was then transferred into a 10-ml disposable column, and the column was washed with 10 bed
volumes of EF-Tu lysis buffer containing 10 mM imidazole, pH 7.5. EF-Tu was finally eluted with EF-Tu lysis buffer containing 30 mM imidazole and dialyzed three times each against 2 liters of EF-Tu storage buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM EDTA, 0.2% sodium
azide, 2 mM phenylmethylsulfonyl fluoride, 10 mM
-mercaptoethanol, 10 µM GDP, and 10%
glycerol). The dialyzed material was divided into 125-µl aliquots,
quick-frozen in liquid nitrogen, and stored at
70 °C. Final EF-Tu
concentration was 3 mg/ml.
-mercaptoethanol. The GlnRS was then dialyzed against
GlnRS storage buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 8 mM
-mercaptoethanol), quick-frozen in 25-µl aliquots, and stored at
70 °C. Final concentration of GlnRS was 11 mg/ml. Aliquots were used only once after thawing, because
of instability of the enzyme upon refreezing or upon storage at higher
temperatures. Expression and purification of His6-tagged methionyl-tRNA synthetase (MetRS) was as described earlier (28).
20 °C. The nucleic acid pellet was washed with 70%
ethanol, air-dried, dissolved in ~10 ml of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), and applied to
a DE52-DEAE-cellulose column (5-ml bed volume) equilibrated with 0.1 M Tris-HCl, pH 7.5. The column was washed with 20 bed
volumes of washing buffer (0.1 M Tris-HCl, pH 7.5, 0.2 M NaCl) and eluted with 0.1 M Tris-HCl
containing 1 M NaCl. Total tRNA from eluate was
ethanol-precipitated, and the pellet was washed twice with ethanol and
dissolved in TE buffer to a final concentration of 100-500
A260/ml. Mutant or wild type
tRNAfMet was purified to homogeneity from the total tRNA by
PAGE, as described (12).
-thiogalactoside and grown for an additional 2 h.
After induction, the cultures were used for assay of
-galactosidase
activity (29) and for the preparation of bacterial extracts and
isolation of total tRNA under acidic conditions (26, 30). Total tRNA
was used for in vivo aminoacylation analysis, and bacterial
extracts were used for assay of
-lactamase (Calbiochem). Activities
of
-galactosidase in extracts were normalized to activities of
-lactamase.
-Lactamase Activity--
Between 3 and 6 µg of
protein in total extracts was taken in 0.9 ml of phosphate buffer Z
(29), and the reaction was started by adding 100 µl of 0.5 mg/ml
nitrocefin at room temperature. The reaction was terminated after 10 min by addition of 10% SDS (110 µl), and the absorbance was read at
486 nm.
-galactosidase and Anti-
-lactamase Antibodies--
Between
10 and 15 µg of protein in total extracts, normalized to
-lactamase levels, were fractionated on an 8% polyacrylamide gel
and transferred to an Immobilon-P membrane (Millipore) by electroblotting. The membrane was probed with polyclonal antibodies against
-lactamase (5 Prime
3 Prime, Inc., Boulder, CO) and
-galactosidase (ICN), using the guidelines provided by the
suppliers. The working dilutions were 1:100,000 for anti-
-lactamase
antibody and 1:20,000 for anti-
-galactosidase antibody. The
immunoblot was probed with secondary horseradish peroxidase-conjugated
goat anti-rabbit IgG (diluted 4,000-fold) and developed for 5 min with a 5-fold dilution of luminol/hydrogen peroxide mixture (1:1, Biolabs) with water, followed by a 15-s exposure to BioMax film (Eastman Kodak
Co.).
(Eq. 1)
For koff measurements, time (in seconds)
was plotted as x axis, and ln(tRNA/tRNAinput),
calculated based on counts/min of the samples, was plotted as
y axis. koff values were then
calculated using the "linear curve fit" option of Kaleidagraph,
using Equation 2,
(Eq. 2)
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ABSTRACT
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Fig. 1.
Cloverleaf structure of E. coli
tRNAfMet and mutants used in this work.
Arrows indicate the sites of mutations. The C50 or A64
mutations were introduced into tRNAs with a U35A36/G72 or U35A36/G72G73
mutant background (A) or a G72 mutant background
(B). The first set of mutant tRNAs is aminoacylated with
glutamine, and the second set is aminoacylated with methionine.
-galactosidase gene. E. coli CA274 (HfrC lacZam
trpEam) was transformed with the pRSV plasmid carrying the mutant
tRNA genes, and cell extracts were used for measurement of
-galactosidase activity. To correct for any possible fluctuations in
plasmid copy number, the
-galactosidase activities were normalized
to the
-lactamase activity encoded in the same pRSV plasmid. As shown before, the U35A36 mutant initiator tRNA is completely inactive as an elongator, whereas the U35A36/G72 and the U35A36/G72G73 mutants
are active in elongation (Table I). Also,
the activity in elongation of the U35A36/G72G73 series of mutants is
higher than that of the corresponding mutants in the U35A36/G72
background. Introduction of C50 or A64 mutations in either the
U35A36/G72 or the U35A36/G72G73 mutant backgrounds substantially
increases
-galactosidase activities. The same results were obtained
in a U1/U35A36 mutant background (data not shown). These results suggest that the U50·G64 wobble base pair restricts the activity of
the E. coli initiator tRNA in elongation. Interestingly, the A64 mutants with a weaker U50:A64 base pair consistently gave a higher
activity in elongation (~5-fold) than the C50 mutants with the
stronger C50:G64 base pair (~3-fold).
Relative activities of mutant tRNAs in suppression in vivo
-galactosidase in cell extracts is
due to increased synthesis of full-length
-galactosidase protein.
Total proteins in cell extracts were fractionated on an SDS-8%
polyacrylamide gel, transferred to Immobilon membrane, and probed with
anti-
-lactamase and anti-
-galactosidase polyclonal antibodies
(Fig. 2). Although the levels of
-lactamase, encoded in the same pRSV plasmid as the mutant tRNA
genes, are, as expected, approximately the same (lanes
1-7), there is more of the full-length
-galactosidase in some
of the extracts compared with others, and the relative intensity of the
bands corresponds to the relative levels of
-galactosidase in cell
extracts.
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Fig. 2.
Immunoblot analysis, using rabbit
anti- -lactamase and
anti-
-galactosidase polyclonal antibodies, of
crude E. coli extracts transformed with mutant tRNAs
shown in Fig. 1A. The truncated
-galactosidase
fragment is produced by chain termination at the site of the amber
mutation. The intensity of the band corresponding to the full-length
-galactosidase reflects the extent of suppression. As expected, this
band and the band corresponding to
-lactamase are absent in extracts
from cells that do not carry the pRSVCAT vector with the mutant tRNA
genes (lane 8).
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Fig. 3.
Northern blot analysis of total RNA isolated
under acidic conditions from E. coli transformed with
plasmids carrying the various mutant tRNAfMet genes.
The blot was probed with 32P-labeled oligonucleotides
complementary to the anticodon stem-loop region of the U35A36 mutant
tRNAfMet and wild type tyrosine tRNA (tRNATyr).
Bands A and B correspond, respectively, to the
uncharged and aminoacylated forms of the mutant tRNAs, and bands
C and D correspond, respectively, to uncharged and
aminoacylated forms of tRNATyr.
Extent of aminoacylation of suppressor tRNAs in vivo
C stem of
the tRNA is protected from cleavage when the aminoacyl-tRNA is in the
form of a ternary complex with EF-Tu·GTP. For the formation of the ternary complex, EF-Tu·GTP and the mutant aminoacyl-tRNAs, labeled with 3H- or 35S-labeled amino acids, were mixed
with various concentrations of His-tagged E. coli
EF-Tu·GTP (serially diluted 2-fold from 1-2 µM down to
1.95-3.9 nM), and the mixture was left on ice for 20 min
to allow complex formation to reach equilibrium. The amount of
aminoacyl-tRNA in the ternary complex was determined by measuring the
amount of 3H or 35S radioactivity remaining
insoluble in 10% trichloroacetic acid after a short treatment (30 s)
with an excess of RNase A. The equilibrium binding curves were then
used to obtain K
View larger version (13K):
[in a new window]
Fig. 4.
Equilibrium binding curves of EF-Tu·GTP
with the various mutant aminoacyl-tRNAs. A, tRNAs
aminoacylated with glutamine; B, tRNAs aminoacylated with
methionine. K
Equilibrium dissociation constants of the various
EF-Tu·GTP·aminoacyl-tRNA ternary complexes measured in Tris-HCl
buffer containing 150 mM NH4Cl
View larger version (14K):
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Fig. 5.
Kinetics of dissociation of
EF-Tu·GTP·aminoacyl-tRNA ternary complex. A, tRNA
aminoacylated with glutamine; B, tRNA aminoacylated with
methionine. Fraction of tRNA, trichloroacetic acid-precipitable
radioactivity remaining after treatment for various times with RNase A
divided by the trichloroacetic acid-precipitable radioactivity at time
0. Aliquots were taken out every 10 min except for the wild type
(W.T.) tRNAfMet (B) in which case it
was every 5 s.
Dissociation rate constants of EF-Tu-GTP-aminoacyl-tRNA ternary
complexes in Tris-HCl buffer containing 150 mM
NH4Cl
View larger version (12K):
[in a new window]
Fig. 6.
Time-dependent cleavage by RNase
A of Met-tRNAfMet, either free ( EF-Tu·GTP) or in the
form of a ternary complex (+EF-Tu·GTP). For the formation of the
ternary complex, the Met-tRNAfMet was incubated with
EF-Tu·GTP on ice for 20 min. The fraction of Met-tRNA,
[35S]Met radioactivity remaining in trichloroacetic
acid-precipitable form after treatment for various times with RNase A
was divided by the trichloroacetic acid precipitable radioactivity at
time 0.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C stem. Interestingly, eukaryotic
initiator tRNAs also have negative determinants at these sites except
that the primary negative determinant is located in the T
C stem and
the secondary negative determinant is the A1:U72 base pair, which is
unique to and is highly conserved in eukaryotic initiator tRNAs (17).
Plant and fungal initiator tRNAs have a bulky
2'-O-phosphoribosyl modification attached to the ribose of
nucleotide 64. This bulky modification protrudes into the minor groove
of the T
C stem and most likely acts as a steric block for the
binding of eEF1 (13-15, 41, 42). In vertebrate initiator tRNAs that
lack the bulky modification, the 50:64 and possibly 51:63 base pairs
act as negative determinants most likely by perturbing the sugar
phosphate backbone in the T
C stem (17). Thus, the negative
determinants for elongation are located at the same sites in bacterial
and in eukaryotic initiator tRNAs. Fig. 7
shows the location of these negative determinants in the initiator
tRNAs in a ribbon diagram of the three-dimensional structure of
tRNA.
View larger version (33K):
[in a new window]
Fig. 7.
Ribbon diagram of three-dimensional structure
of tRNA highlighting the negative determinants for elongation in
E. coli and in eukaryotic initiator tRNAs.
Does the secondary negative determinant in the TC stem of E. coli initiator tRNA affect elongation activity of the tRNA by modulating the binding affinity of the aminoacyl-tRNA to EF-Tu·GTP? Binding assays show that mutation of the U50·G64 wobble base pair to
C50:G64 or U50:A64 increases only marginally the binding affinity of
the aminoacyl-tRNAs to EF-Tu·GTP. The koff
values of the ternary complexes formed between EF-Tu·GTP and the
mutant aminoacyl-tRNAs suggest a more significant increase,
2.4-2.6-fold, in stability of the ternary complexes formed with the
C50 and A64 mutant aminoacyl-tRNAs (Table IV, top half). Whether these
small improvements in binding affinity for EF-Tu·GTP and the
stabilities of the EF-Tu·GTP·aminoacyl-tRNA ternary complexes are
sufficient to account for the increased activities of these mutant
tRNAs as amber suppressors in vivo (Table I) or whether the
increased activity is also due to a better accommodation of the A64 and
C50 mutant tRNAs into the ribosomal A site is not known. It is
important to note, however, that 2-3-fold increases in
vitro in EF-Tu·GTP binding affinity of the Su+7 tRNA
aminoacylated with glutamine over that aminoacylated with tryptophan
(43) results in selection in vivo of glutamine over tryptophan by a factor of 9 (44, 45).
A striking result of our work is the very large effect of the amino
acid attached to the tRNA on EF-Tu binding affinity of wild type and
mutant initiator tRNAs. The binding of the wild type tRNA aminoacylated
with methionine is so unstable that it was not possible to obtain an
accurate estimate of K4
s
1 (Table IV) corresponding to a half-life of 15 s,
the ternary complex formed with the U35A36 mutant
Gln-tRNAfMet had a dissociation rate constant of 6.21 × 10
4 s
1 corresponding to a half-life of
17 min. Because the only difference between these two tRNAs is in the
anticodon sequence and EF-Tu is known not to interact with the
anticodon (9), the difference in binding affinities and stability of
the ternary complexes is due to the different amino acids attached to
the tRNA. Knowlton and Yarus (43) were the first to show that
Su+7 tRNA aminoacylated with glutamine bound to EF-Tu·GTP
with a K
The binding (K
The strong effect of methionine in modulating the binding affinity of
Met-tRNAfMet for EF-Tu highlights yet another important
reason why methionine is the best amino acid for the initiation of
protein synthesis in E. coli. Initiation requires
formylation of the initiator Met-tRNA to fMet-tRNA by MTF. The formyl
group then acts as a positive determinant for IF2. We and others
(47-49) have shown that MTF prefers methionine over all the other
amino acids tested. Similarly, IF2 also prefers formylmethionine over
several other formyl amino acids tested (51). Our finding that EF-Tu
binds extremely poorly to the wild type Met-tRNAfMet but
binds quite well to the U35A36 mutant Gln-tRNAfMet provides
yet another role for methionine in minimizing the affinity of the
initiator Met-tRNAfMet for EF-Tu. This allows MTF, which is
present in extremely small amounts in E. coli (57), to
compete for Met-tRNAfMet with EF-Tu, which is the most
abundant protein in E. coli (58). Finally, although
fMet-tRNAfMet is not a substrate for peptidyl-tRNA
hydrolase (59), Varshney and co-workers (54) have shown that the U35A36
mutant fGln-tRNAfMet is a substrate for peptidyl-tRNA
hydrolase. Thus, methionine plays yet another important role in
ensuring the availability of fMet-tRNA for initiation by preventing its
hydrolysis by peptidyl-tRNA hydrolase.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Linda Spremulli for the strain for overproducing EF-Tu, Dr. Alexey Wolfson for advice on assays for EF-Tu binding, and Dr. Caroline Köhrer for help in the design of figures for electronic submission. We thank Annmarie McInnis for the continued enthusiasm and for patience and care in the preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant R37GM17151 from the National Institutes of Health.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.
Present address: Dept. of Microbiology and Cell Biology, Indian
Institute of Science, Bangalore 560012, India.
§ To whom correspondence should be addressed: Dept. of Biology, Rm. 68-671, Massachusetts Institute of Technology, Cambridge, MA 02139. Tel.: 617-253-4702; Fax: 617-252-1556; E-mail: bhandary@mit.edu.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M212890200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EF, elongation factors; GlnRS, glutaminyl-tRNA synthetase; MetRS, methionyl-tRNA synthetase; MTF, methionyl-tRNA formyltransferase.
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