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From the
In Escherichia coli, lysyl-tRNA synthetase activity is
encoded by either a constitutive lysS gene or an inducible
one, lysU. The two corresponding enzymes could be purified at
homogeneity from a
Homogeneous LysU
and LysS were also compared in Ap
In a cell, the covalent attachment of one amino acid to its
cognate tRNAs is, in principle, performed by a single aminoacyl-tRNA
synthetase. A rare exception to this rule is the case of Escherichia coli lysyl-tRNA synthetase (LysRS),
lysU expression is induced (i) by the addition of alanine,
leucine, or leucine-containing dipeptides in the growth
medium(3, 4, 5) or (ii) by exposure to either
anaerobiosis(6) , high temperature(7) , or low external
pH (8, 9) conditions. Recently, lysU expression
was shown to be under the negative control of the leucine-responsive
regulatory protein (Lrp)(10, 11, 12) , a global
regulatory factor that would participate to the adaptation of E.
coli to its environment in the host intestinal tract(13) .
The effect of this regulatory protein on lysU expression is
antagonized by the presence of leucine(14) .
The lysS gene makes part of a dicistronic transcriptional unit comprising
also prfB(15) . The latter gene encodes the peptide
chain release factor specific of stop codons UAA and UGA. The
translation of prfB is tightly autoregulated by a mechanism
requiring a +1 frameshift at a UGA codon(16) . Because of
the proximity between the stop codon of the prfB open reading
frame and the downstream initiator codon of lysS(15) ,
the expression of lysS is likely to be influenced by the
translation of prfB. Besides, lysS does not respond
to the above physiological conditions which induce lysU expression(1, 17) .
Gene disruption experiments
showed that each of the lysS and lysU genes is
dispensable for the growth of the bacterium.
The sequences of the two E.
coli LysRS species share 88.5% identity, with 447 identical amino
acids out of a total of 505 (Ref. 2 and corrected sequence of LysU
under EMBL/GenBank
In the present study, advantage was taken of
the availability of
Polyacrylamide gel electrophoresis analyses were performed with the
Pharmacia Phast gel apparatus under the conditions recommended by the
supplier.
Hydroxylapatite chromatography of the
dialyzed proteins was performed through a column of 2.6
At this stage, the LysS protein was
already more than 95% pure, as judged from sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis. In the case of
LysU, further chromatographic steps were required; after ammonium
sulfate precipitation (80%) and subsequent dialysis against buffer A,
the protein sample containing LysU (20 ml) was applied onto a
DEAE-Sephadex column (1.3
For
storage at -20 °C, the enzymes (10 mg/ml) were dialyzed
against 20 mM Tris-HCl buffer (pH 7.8) containing 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and 60% glycerol.
In the
case of the
Enzyme specific
activity was calculated using protein concentrations determined by the
Bio-Rad protein assay, with bovine serum albumin as the standard. In
the cases of pure LysS or LysU, concentrations were measured using a UV
absorption coefficient of 0.5 A
Initial
rates of the ATP-PP
The synthesis of Ap
All Michaelian parameters were derived from iterative
nonlinear fits of the theoretical rate equations to the experimental
values, using the Levenberg-Marquardt algorithm(27) .
To purify LysS or LysU, a
chromatographic step on hydroxylapatite was used. At this stage, a
broad and asymmetrical peak of LysS activity suggested several
subspecies of the enzyme (Fig. 1). Such a behavior was
reminiscent of previous observations that E. coli LysRS eluted
as multiple peaks on
hydroxylapatite(29, 30, 31, 32) . To
verify that the properties of the overproduced enzyme were identical to
those of LysS of chromosomal origin, a control experiment was performed
with the
These purification procedures yielded 140 and 25 mg of LysS and
LysU, respectively, from 9 g of wet cells ().
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy the experimental data could be fitted to the following
relationship,
On-line formulae not verified for accuracy where v is the initial rate of exchange,
[E
Finite
values of K
The measured k
LysU sustained a rate of Ap
The reason for the occurrence of two LysRS genes in E.
coli has to be searched for at the regulatory or the functional
level. A first possibility is that the juxtaposition of two genes
results in a finer regulation of cellular LysRS concentration. For
instance, the coordination of lysS and prfB expressions might become harmful in some cases and the switch-on
of a second LysRS gene would offer the possibility to escape the
coupling between lysS and prfB. Alternatively, the
addition of two LysRS genes can provide a functional advantage,
provided the characteristics of each synthetase are adapted to
different compositions of the cytoplasm. In such a case, however, some
discrepancy should be recognizable when comparing the biochemical
properties of LysS and LysU.
This study indicates that the greatest
difference between LysS and LysU is at the level of their affinities
for lysine, the dissociation constant of the LysU-lysine complex being
8-fold smaller than that of the LysS-lysine complex. In contrast, the
binding parameters of ATP and purified tRNA
To evaluate the advantage of having two LysRS
species with distinct K
To our knowledge, little
information is available on the cadaverine concentration in E. coli cells. Igarashi et al. measured a cadaverine amount of
130 nmol/g of wet cells in the case of E. coli B cells grown
in minimal medium without lysine(42) . With a mutant strain
lacking the biosynthetic arginine decarboxylase (43) grown in another
minimal medium without lysine, Kashiwagi and Igarashi found 2.3 nmol of
cadaverine/mg of protein. These values, which correspond to
200-700 µM cellular cadaverine, are likely to
increase under conditions capable of inducing lysine decarboxylase
activity. Under appropriate conditions, the activity of this enzyme
increases in the cell by a factor of up to 3000 (44), and lysine
decarboxylase may represent nearly 2% of the total E. coli proteins(45) . Consequently, even if the major part of the
produced cadaverine were excreted (or sequestered by binding to
membrane phospholipids or nucleic acids), it would seem reasonable to
imagine that, upon lysine decarboxylase induction, free cellular
cadaverine concentration might reach values exceeding the K
Interestingly, the regulation of lysU shares common features with that of the cadBA operon: (i) lysU and cadBA are induced by
anaerobiosis or by low external pH(6, 8, 46) ;
(ii) the induction of these genes occurs only when bacteria are grown
in rich medium(6, 44) ; (iii) the expressions of both lysU and cadA genes are enhanced in mutants of hns(47, 48) and in mutants of cadR(18, 44) , a gene suspected to be identical
to the lysP gene encoding lysine permease(49) .
Moreover, (iv) homologous DNA sequences are found upstream of cadB and lysU(6) in regions shown to be important for
the regulations of these genes(11, 50) . In turn, cadBA expression is induced by the addition of external lysine
while lysU expression is not(45, 51) . The
latter behavior is in agreement with the idea that, in the presence of
a high supply of lysine, the derepression of lysU would become
unnecessary. Although cadaverine will accumulate following cadBA induction, a high cellular lysine concentration would protect
LysRS activity against inhibition by the polyamine.
If the two LysRS
species of E. coli functionally account for various
physiological conditions, the question arises why two genes have been
selected during evolution to produce two synthetases, rather than
post-translational modifications of a single gene product. In truth,
evolution may have favored also the latter mechanism to multiply the
diversity of LysRS species in E. coli. As shown here and
already often noticed before, LysRSs appear as multiple subspecies upon
hydroxylapatite chromatography (29-32). Two-dimensional gel
electrophoresis experiments also revealed that the products of lysS and even of lysU may occur under several forms in
vivo(1) . The biological significance of such variants, and
the nature of the corresponding maturations remain mysterious. However,
it reminds us the behavior of tryptophanyl-tRNA synthetase, which is
also recovered as two distinct subspecies from hydroxylapatite
chromatography(52) , and those of threonyl- and glutaminyl-tRNA
synthetases, which are partly phosphorylated in vivo through a
mechanism involving heat-shock proteins DnaJ and DnaK(53) .
These cases which escape the rule ``one amino acid-one
aminoacyl-tRNA synthetase'' are likely to reflect the capability
of E. coli to adapt the activity of the synthetases to its
environment in a yet unsuspected manner.
We gratefully acknowledge F. Dardel for fruitful
advice.
Volume 270,
Number 24,
Issue of June 16, pp. 14439-14444, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
lysU and a lysS strain,
respectively. Comparison of the pure enzymes, LysS and LysU, indicates
that, in the presence of saturating substrates, LysS is about twice
more active than LysU in the ATP-PP
exchange as well as in
the tRNA aminoacylation reaction. Moreover, the
dissociation constant of the LysU-lysine complex is 8-fold smaller than
that of the LysS-lysine complex. In agreement with this difference, the
activity of LysU is less sensitive than that of LysS to the addition of
cadaverine, a decarboxylation product of lysine and a competitive
inhibitor of lysine binding to its synthetase. This observation points
to a possible useful role of LysU, under physiological conditions
causing cadaverine accumulation in the bacterium. Remarkably, these
conditions also induce lysU expression.
A synthesis. LysU is only
2-fold more active than LysS in the production of this dinucleotide.
This makes unlikely that the heat-inducible LysU species could be
preferentially involved in the accumulation of ApA inside
stressed Escherichia coli cells. This conclusion could be
strengthened by determining the concentrations of ApN (N
= A, C, G, or U) in a lysU as well as in a lysU strain, before and after a 1-h
temperature shift at 48 °C. The measured concentration values were
the same in both strains.
()which occurs as two distinct
species(1, 2) . The corresponding genes, lysS and lysU, are submitted to different regulations.
lysU cells
do not display a clear phenotype. Clark and Neidhardt reported that the
growth of a lysU strain at 44 °C is slightly slower
than that of the isogenic lysU strain(18) . However, this difference could not be
reproduced in other genetic contexts(6, 19) . Disruption
of lysS renders the cell
cold-sensitive(6, 20) . This behavior can be explained
by the low expression of lysU at growth temperatures below 37
°C. Accordingly, transformation of a
lysS strain with
a plasmid overexpressing lysU is enough to cure the
cold-sensitive phenotype(6) .
accession number X16542). The homology is,
however, smaller than in the case of other E. coli isoenzymes.
For instance, the two EF-Tu species or the two glutamate decarboxylases
display 99.7 and 98.9% identity, respectively(21, 22) .
Therefore, the question arises to know whether the two E. coli LysRSs may be functionally distinct. One possibility is that the lysU product has features directly or indirectly useful for
the cell to adapt to extreme conditions such as high temperatures, high
pH, or anaerobiosis.
lysS and lysU strains
to prepare samples of either the LysU or the LysS protein and to
compare their kinetic properties. The most noticeable difference is an
affinity of LysU for lysine nearly 8-fold higher than that of the LysS
species. In agreement with this difference, LysU activity is relatively
less sensitive than the LysS one to the presence of cadaverine, an
inhibitor of lysine binding. The latter polyamine is produced at the
expense of lysine in several stressing cellular conditions. Remarkably,
most of these conditions induce lysU expression too.
Materials
L-[
C]Lysine
(11.1 GBq/mmol) was from Amersham, [P]PP
(300 GBq/mmol) was from DuPont NEN. ApA was from
Boehringer, cadaverine (1,5-diaminopentane) was from Aldrich.
Unfractionated E. coli tRNA was from Boehringer, and purified E. coli tRNA (1000 pmol of lysine
acceptance/A
unit) from Sigma. Hydroxylapatite
was from Bio-Rad and DEAE-Sephadex A50 from Pharmacia.
Bacterial Strains
The bacterial strains used in
this study included XA103 (F
(lac-pro) gyrA rpoB metB argE(Am) ara supF)(23) , PAL2103UKTR (F
(lac-pro) gyrA rpoB metB argE(Am) ara supF lysU::kan
srl-300::Tn10 recA56) and PAL3103SKTR
(F
(lac-pro) gyrA rpoB
metB argE(Am) ara supF lysS::kan
srl-300::Tn10 recA56)(6) . For the
purifications of LysS and LysU, strain PAL2103UKTR transformed by
plasmid pXLysKS1 (lysS) (2) and strain
PAL3103SKTR transformed by pXLys5 (lysU
) (24) were used, respectively.
Purification of LysS and LysU from Overproducing
Strains
All steps were performed at 4 °C in buffers
systematically containing 10 mM 2-mercaptoethanol and 0.1
mM EDTA. The cells from an overnight culture were harvested by
centrifugation during 45 min at 5000 g. The pellet was
suspended in 20 mM Tris-HCl buffer (pH 7.8) at a cell density
of 0.15 g (wet weight)/ml of buffer and sonicated for 10 min. Cell
debris were removed by centrifugation at 10,000
g for
20 min. Then, streptomycin sulfate was added to the supernatant at a
final concentration of 27 mg/ml. After centrifugation of the sample for
20 min at 10,000
g, the supernatant was brought to 80%
ammonium sulfate saturation and centrifuged for 30 min at 10,000
g. The protein pellets were dissolved in 20 mM potassium phosphate buffer (pH 6.75) (buffer A) and dialyzed
against the same buffer.
19 cm
equilibrated in buffer A. After a 300-ml wash with buffer A, elution
was carried out at a flow rate of 30 ml/h using a linear gradient of
potassium phosphate concentration (2.5 liters, from 20 to 300
mM). Fractions displaying LysRS activity were pooled and
dialyzed against buffer A.
3.8 cm; 4.2 ml/h) equilibrated in 50
mM potassium phosphate (pH 6.75) (buffer B). The column was
washed with 5 ml of buffer B containing 100 mM KCl. Elution
was performed with a linear gradient of KCl concentration in buffer B
(125 ml, from 100 to 500 mM KCl). Fractions exhibiting LysU
activity were pooled (20 ml) and directly loaded onto a hydroxylapatite
column (2
4.8 cm; 6 ml/h) equilibrated in buffer A. The column
was washed with buffer A, and LysU was eluted by a linear gradient of
potassium phosphate (600 ml, from 50 to 300 mM). Active
fractions were pooled (100 ml) and dialyzed against buffer A.
Characterization of the LysRS Subspecies in Various
Strains
To analyze the LysRS subspecies produced by the
lysS and lysU strains, crude extract
preparation from 250-ml cultures, precipitation of nucleic acids with
streptomycin sulfate and precipitation of proteins with ammonium
sulfate were performed as described above. A hydroxylapatite
chromatography was further performed on a 1.7 4-cm column
equilibrated in buffer A. After a 50-ml wash with buffer A, elution was
insured at a flow rate of 10 ml/h using a linear gradient of potassium
phosphate concentration (225 ml, from 20 to 300 mM).
lysU extracts, the hydroxylapatite
chromatography produced three peaks of LysRS activity each of which was
dialyzed against 20 mM potassium phosphate buffer (pH 8.2) and
applied onto a DEAE-Sephadex A50 column (0.3 9 cm) equilibrated
in 20 mM potassium phosphate buffer (pH 8.2). Elution was
performed at a flow rate of 0.5 ml/h using a linear gradient of
potassium phosphate concentration (10 ml, from 20 to 500 mM).
Enzymatic Assays
Unless otherwise stated, initial
rates of tRNA aminoacylation were measured at 37 °C in 100 µl
of 20 mM Tris-HCl buffer (pH 7.8), 150 mM KCl, 2
mM ATP, 0.1 mM EDTA, 7 mM MgCl
,
5 kBq L-[C]lysine, 30 µM lysine, and 150 µM unfractionated E. coli tRNA. After incubation in the presence of catalytic amounts of
enzyme, tRNA was precipitated with trichloroacetic acid, filtered, and
counted for incorporated C as described
previously(25) . One unit of LysRS activity is the amount of
enzyme producing 1 µmol of lysyl-tRNA/min.
units
mg
ml(24) .
exchange reaction were measured at 25
°C in 100 µl of 20 mM Tris-HCl buffer (pH 7.8), and
0.1 mM EDTA, containing 3-15 kBq
[P]PP
plus various concentrations of
ATP, lysine, and unlabeled PP. MgCl in the
assay systematically exceeded by 3 mM the sum of the ATP and
PP concentrations. The reaction was initiated by the
addition of catalytic amounts of the LysRS under study. Labeled ATP was
adsorbed on charcoal, filtered, and counted as described
previously(26) .
A was assayed
at 37 °C by bioluminescence(24) . The incubation mixture
included 20 mM Tris-HCl buffer (pH 7.8), 150 mM KCl,
7 mM MgCl, 5 mM ATP, 100 µM lysine, 150 µM ZnCl, 0.04 mg/ml yeast
pyrophosphatase (from Boehringer) and catalytic amounts of the LysRS
under study.
Purification of LysS and LysU
To avoid any
cross-contamination between LysS and LysU proteins, LysS was purified
from a lysU null mutant overexpressing lysS, whereas
LysU was purified from a lysS null mutant overexpressing lysU. Strains were grown overnight at 37 °C in 2 liters of
2 TY medium (28) containing 100 µg/ml ampicillin. In
these conditions, the two above strains overproduced LysRS activity
200- and 36-fold, respectively, as compared with the parental strain
XA103 (lysS
lysU
)(23) .
lysU strain PAL2103UKTR. With this strain, three
distinct peaks of LysRS activity were observed (Fig. 1), which
were separately pooled and further purified on DEAE-Sephadex columns.
The three enzymes subspecies were indistinguishable according to their K values for lysine (at ATP and PP
concentrations of 2 mM and 0.15 mM,
respectively) and for ATP (2 mM lysine and 0.15 mM PP) in the ATP-PP exchange reaction.
Consequently, a single pool was made from the broad asymmetrical peak
obtained with the overproduced LysS enzyme. The pooled protein behaved
homogeneous according to SDS-polyacrylamide gel electrophoresis
analysis and showed K values for lysine
and ATP identical to those measured with the three subspecies of LysS
of chromosomal origin.
Figure 1:
Hydroxylapatite
chromatography of extracts of strain PAL2103UKTR (lysU) (A) and of the same strain transformed by plasmid pXLysKS1
harboring lysS (B). Columns were eluted using a
linear gradient of potassium phosphate concentration from 20 to 300
mM (pH 6.75). The A of the column
effluent was monitored (
). Prior to tRNA aminoacylation
measurements the fractions of the profiles A and B were diluted 80- and
20,000-fold, respectively (
).
Overproduced LysU eluted from the
hydroxylapatite column at 100 mM phosphate, a concentration
significantly lower than that required to elute LysS (140 mM).
The activity profile was symmetrical. A control experiment with a
lysS strain showed a single LysU species of chromosomal
origin also eluting at 100 mM phosphate (data not shown). To
obtain homogeneous enzyme, the sample of overproduced LysU had to be
further purified on DEAE-Sephadex and once more on hydroxylapatite.
ATP-PP
Initial rates of LysS and LysU in the isotopic
ATP-[Exchange
ReactionP]PP
exchange reaction were
compared as a function of lysine, ATP, and PP concentrations. In a first set of experiments, apparent K values for lysine (K
) were measured at
various nonsaturating ATP concentrations, while PP
concentration was fixed equal to 150 µM. Whatever
the LysRS species and the ATP concentration studied, Michaelian
kinetics were always observed. The apparent k and K
values
depended on the ATP concentration so that double-reciprocal plots
produced lines which intersected on the left of the ordinate axis. The results with LysS are shown in Fig. 2. When PP
concentration was varied in the
presence of fixed concentrations of lysine (2 mM) and ATP (2
mM), Michaelian kinetics were obtained again with each enzyme,
yielding K
Figure 2:
Double-reciprocal plot of the initial rate
of the ATP-PP exchange catalyzed by LysS enzyme versus lysine concentration in the assay. The initial rates were measured
at ATP concentrations of 0.02 mM (
), 0.1 mM (), 0.5 mM (
), 1 mM (
), or 2
mM (). The straight lines were calculated
with parameters obtained through the fitting of the entire set of data
to the reaction scheme. For the sake of clarity, only shown is the
portion of the plot corresponding to the highest lysine concentrations
and to the highest initial rates.
According to the following reaction scheme ()(26) ,
] is the total enzyme concentration, k
is the catalytic constant of the reaction, and K
=
[E][Lys]/[E:Lys], C
=
[E:Lys][ATP]/[X], C
=
[E:Lys
AMP][PP
]/[X], K =
[E][ATP]/[E:ATP], C
=
[E:ATP][Lys]/[X]
= C
K
/K
.
and K
could
be deduced (), thereby suggesting random rather than
ordered binding of ATP and lysine to the enzyme. A mechanism where
lysine binds first would have produced an infinite value of K
, and the lines in Fig. 2would have
intersected on the ordinate axis. Similarly, a mechanism where ATP
binds first would have produced an infinite value of K
. However, a mechanism where one out of the two
substrates would form an abortive complex with the enzyme cannot be
ruled out since it would also be compatible with the above rate law
(33).
and K
values of LysU were respectively 2.3- and 1.6-fold lower than
those of LysS. In contrast, the dissociation constant of lysine
complexed to LysU in the absence of ATP (K
) was
nearly 8-fold smaller than that of the lysine-LysS complex. The value
of C
, which corresponds to the K
value for lysine at saturation of both
ATP and PP
, was also smaller by a factor of 4 in the case
of LysU as compared with its value in the case of LysS ().
Aminoacylation of tRNA
In another
set of experiments, initial rates of tRNA aminoacylation
by either LysS or LysU were compared. A Michaelian behavior was
observed in the case of each LysRS. The K
value of LysU for lysine (0.7 µM), measured at
a fixed unfractionated tRNA concentration of 150 µM, was
significantly lower than the K
value of
LysS (4.5 µM). At 30 µM lysine, the K
value for purified tRNA
was 2.6-fold higher with LysU than with LysS ().
Ap
ApA SynthesisA is a
representative member of a family of dinucleoside polyphosphates
(ApN, where N = A, C, G, or U and n = 3 or 4) detectable in most if not all living cells (34) and known to be produced in vitro as well as in vivo by various aminoacyl-tRNA synthetases, including the
LysRSs(35) . Because dinucleoside polyphosphates strongly
accumulate in response to various stress conditions including heat
shock(36) , we searched for whether the heat-inducible LysU
protein could be more efficient than LysS in promoting Ap
A
synthesis.
A synthesis only
2.1-fold higher than that with LysS (). Therefore, it is
unlikely that the LysU species is preferentially involved in the
ApN accumulation observed during a heat shock response. To
reinforce this conclusion, the ApN contents of strains
PAL2103UKTR (lysU) and XA103 (lysU) were compared before and after a
temperature shift. Bacteria were grown aerobically in LB medium, at 37
°C. When the optical density of the culture reached 0.3 at 650 nm,
Ap
N were extracted from an aliquot of the cultures and
assayed. The ApN concentration amounted to 2.8 and 3
µM in the lysU and lysU strains, respectively. The remaining part of the culture was
shifted from 37 to 48 °C. After 60 min at 48 °C, the
Ap
N concentration in the lysU strain (11
µM) remained similar to that in the lysU strain (11 µM).
Inhibition by Cadaverine
In principle, a LysU
protein having a higher affinity for lysine than that of LysS might be
beneficial to the cell when intracellular lysine concentration becomes
limiting. Alternatively, LysU might be useful to resist inhibition by a
lysine analog. One such naturally occurring analog is cadaverine.
Therefore, the initial rate of the ATP-PP exchange was
measured at fixed concentrations of ATP (2 mM) and PP (2 mM), in the presence of various concentrations of
lysine and cadaverine. In the case of both LysRSs, cadaverine behaved
as a competitive inhibitor of lysine. The corresponding K values toward LysS and LysU were
similar (53 and 73 µM, respectively). Consequently, at a
saturating concentration of lysine, LysU was less sensitive to the
inhibition by cadaverine than LysS, because of the lower K
value for lysine of the former enzyme.
Thus, at lysine concentrations greater than 7 µM, 50%
inhibition of LysU activity required a cadaverine concentration at
least six times greater than that causing 50% inhibition of LysS
activity.
Thermostability
Because lysU gene
expression accompanies the adaptation of the bacterium to thermal
transition, it was of interest to compare the thermostabilities of LysU
and LysS. This measurement was made in a 20 mM Tris-HCl buffer
(pH 7.8) containing 7 mM MgCl, 0.1 mM EDTA, 2 mM ATP, 30 µML-lysine, and
150 µM of unfractionated E. coli tRNA. After
incubation at 42 °C for various times, aliquots (100 µl) were
removed from the assay and the initial velocity of tRNA aminoacylation
was determined in a standard aminoacylation mixture. The kinetics of
inactivation at 42 °C followed exponential curves (Fig. 3)
with half-life times of LysS and LysU activities of 62 and 141 s,
respectively, in agreement with previous results indicating that LysU
was more resistant to thermal inactivation than
LysS(32, 37) .
Figure 3:
Comparison of the thermostabilities of
LysS () and LysU (
). Residual tRNA aminoacylation activity
(expressed as percentage of the activity at time 0) is plotted as a
function of the time of incubation. Enzymes (0.2 µM) were
incubated at 42 °C in the presence of 20 mM Tris-HCl (pH
7.8), 0.1 mM EDTA, 7 mM MgCl
, 2 mM ATP, 30 µML-lysine, and 150 µM unfractionated E. coli tRNA.
are similar
with the two synthetases. Although only one tRNA
isoacceptor has yet been described(38) , in agreement with
the sequence identity of the three E. coli tRNA
genes(39) , it cannot be excluded that, under particular
growth conditions, post-transcriptional modifications may render
tRNA
able to distinguish between LysS and LysU. For
instance, the tRNA
molecule has been shown to incorporate
selenium when the growth medium contains micromolar amounts of this
element(40) .
values for
lysine, it is interesting to involve cadaverine, a polyamine produced
from lysine by a decarboxylase. The cadA gene encoding lysine
decarboxylase forms an operon with cadB, a gene corresponding
to an antiporter protein capable of importing one lysine molecule while
it excretes one cadaverine molecule(41) . The coupled actions of
the cadA and cadB products is believed to be involved
in the adaptation of the bacterium to low external pH, since the
conversion of each lysine molecule to cadaverine consumes one
proton(41) . However, this mechanism may transiently both
decrease intracellular lysine and increase internal cadaverine. Since
cadaverine behaves as a competitive inhibitor of lysine binding to
LysRS (see ``Results''), a stimulated production of LysU, the
activity of which is less sensitive to cadaverine than that of the
constitutive LysS, could be useful.
value of cadaverine toward any LysRS
species (
50 µM).
Table: Purification of LysS and LysU proteins
Table: Kinetic constants of LysS and LysU in the
ATP-PP exchange, tRNA aminoacylation, and
Ap
A synthesis reactions
A and Other Dinucleoside Polyphosphates (McLennan, A. G., ed) pp. 29-61, CRC Press, Boca Raton, FL
A and Other Dinucleoside Polyphosphates (McLennan, A. G., ed) pp. 63-79, CRC Press, Boca Raton, FL
A and Other Dinucleoside Polyphosphates (McLennan, A. G., ed) pp. 135-149, CRC Press, Boca Raton, FL
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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B. C. Jester, J. D. Levengood, H. Roy, M. Ibba, and K. M. Devine Nonorthologous replacement of lysyl-tRNA synthetase prevents addition of lysine analogues to the genetic code PNAS, November 25, 2003; 100(24): 14351 - 14356. [Abstract] [Full Text] [PDF]
|
|
|
|
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|
C. Francklyn tRNA synthetase paralogs: Evolutionary links in the transition from tRNA-dependent amino acid biosynthesis to de novo biosynthesis PNAS, August 19, 2003; 100(17): 9650 - 9652. [Full Text] [PDF]
|
|
|
|
|
|
A. Brevet, J. Chen, S. Commans, C. Lazennec, S. Blanquet, and P. Plateau Anticodon Recognition in Evolution: SWITCHING tRNA SPECIFICITY OF AN AMINOACYL-tRNA SYNTHETASE BY SITE-DIRECTED PEPTIDE TRANSPLANTATION J. Biol. Chem., August 15, 2003; 278(33): 30927 - 30935. [Abstract] [Full Text] [PDF]
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 |
|
 M. Fromant, M.-L. Ferri-Fioni, P. Plateau, and S. Blanquet Peptidyl-tRNA hydrolase from Sulfolobus solfataricus Nucleic Acids Res., June 15, 2003; 31(12): 3227 - 3235. [Abstract] [Full Text] [PDF]
|
|
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G. Rosas-Sandoval, A. Ambrogelly, J. Rinehart, D. Wei, L. R. Cruz-Vera, D. E. Graham, K. O. Stetter, G. Guarneros, and D. Soll Orthologs of a novel archaeal and of the bacterial peptidyl-tRNA hydrolase are nonessential in yeast PNAS, December 24, 2002; 99(26): 16707 - 16712. [Abstract] [Full Text] [PDF]
|
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M. Sissler, C. Delorme, J. Bond, S. D. Ehrlich, P. Renault, and C. Francklyn An aminoacyl-tRNA synthetase paralog with a catalytic role in histidine biosynthesis PNAS, August 3, 1999; 96(16): 8985 - 8990. [Abstract] [Full Text] [PDF]
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M. Ibba, H. C. Losey, Y. Kawarabayasi, H. Kikuchi, S. Bunjun, and D. Soll Substrate recognition by class I lysyl-tRNA synthetases: A molecular basis for gene displacement PNAS, January 19, 1999; 96(2): 418 - 423. [Abstract] [Full Text] [PDF]
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J.-M. Guillon, S. Heiss, J. Soutourina, Y. Mechulam, S. Laalami, M. Grunberg-Manago, and S. Blanquet Interplay of Methionine tRNAs with Translation Elongation Factor Tu and Translation Initiation Factor 2in Escherichia coli J. Biol. Chem., September 13, 1996; 271(37): 22321 - 22325. [Abstract] [Full Text] [PDF]
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