tmRNA from
Thermus thermophilus
Interaction with alanyl-tRNA synthetase and elongation factor Tu
Victor G. Stepanov and Jens Nyborg
Institute of Molecular and Structural Biology, University of Aarhus, Denmark
The interaction of a Thermus thermophilus tmRNA tran-
script with alanyl-tRNA synthetase and elongation factor
Tu has been studied. The synthetic tmRNA was found to be
stable up to 70 °C. The thermal optimum of tmRNA
alanylation was determined to be around 50 °C. At 50 °C,
tmRNA transcript was aminoacylated by alanyl-tRNA
synthetase with 5.9 times lower efficiency (k
cat
/K
m
)
than tRNA
Ala
, primarily because of the difference in
turnover numbers (k
cat
). Studies on EF-Tu protection of
Ala$tmRNA against alkaline hydrolysis revealed the
existence of at least two different binding sites for EF-Tu
on charged tmRNA. The possible nature of these binding
sites is discussed.
Keywords:tmRNA;elevatedtemperatures;alanyl-tRNA
synthetase; EF-Tu.
The transfer-messenger RNA (tmRNA) is a small stable
bacterial RNA that is an object of considerable interest
because of its obvious structural and functional dualism.

of proteins. The identity determinants of the tRNA-like
module of the tmRNA are equivalent to those of tRNA
Ala
,
so that tmRNA can be charged with alanine by alanyl-
tRNA synthetase [4,5]. EF-Tu*GTP has been shown to
form a complex with alanylated tmRNA, in which the ester
bond between the alanyl residue and the 3¢-terminal
adenosine of tmRNA is protected against hydrolysis as in
the canonical ternary complex between EF-Tu, GTP and
aminoacyl-tRNA [5,6]. Two other proteins, S1 and SmpB,
are indispensable for the proper interaction of the tmRNA
with the ribosome. S1 binds near the mRNA-like module
and probably assists the entrance of the tag-encoding
tmRNA part into the ribosome [7]. SmpB can bind to the
tRNA-mimicking domain simultaneously with EF-Tu and
presumably stabilizes the active conformation of this
tmRNA region [8]. The significant stimulative effect of
SmpB on the efficiency of tmRNA aminoacylation [9]
makes it likely that this protein is an integral part of a
tmRNA-based ribosome rescue complex in vivo. In contrast,
SmpB was found to inhibit the tRNA
Ala
aminoacylation
reaction [8]. Some other proteins, RNase R, SAF and
phosphoribosyl phosphorylase, were also observed to form
tight complexes with tmRNA, but their roles and the
location of their binding sites on tmRNA remain elusive
[10]. Thus, it is evident that tmRNA performance on the
ribosome requires the assistance of numerous protein

m
) becomes higher under the
influence of SmpB, or when synthesized Ala$tmRNA is
trapped in a complex with EF-Tu*GTP and thus stabilized.
As a result, the plateau of the tmRNA aminoacylation
reaction could be increased to the biologically relevant level
in the presence of these proteins.
The major part of the above-mentioned features of the
trans-translation mechanism has been revealed in experi-
ments with Escherichia coli tmRNA and proteins. Studies
on tmRNAs from other sources have been sporadic and
have addressed only a limited number of special issues. In
the context of our studies on the translation apparatus of
Thermus species, we aimed to investigate Ala$tmRNA
synthesis with alanyl-tRNA synthetase and its binding to
elongation factor Tu. Taking into account the increased
lability of the alanyl ester bond at high temperatures [12],
the thermophile should encounter (and somehow overcome)
the intense spontaneous deacylation of the Ala$tmRNA. A
hot environment may in this way imprint the character
of the specific interactions between the macromolecules
involved in trans-translation in T. thermophilus.Herewe
describe assays on thermophilic tmRNA, alanyl-tRNA
synthetase and EF-Tu, related to their activity in the trans-
translation reaction at elevated temperatures.
Materials and methods
Chemicals, RNAs and proteins
L
-[2,3–
3

Biolabs.
Construction of a recombinant plasmid harbouring
the tmRNA gene
The wild-type tmRNA gene, ssrA, was amplified from
T. thermophilus HB8 genomic DNA by a Taq DNA
polymerase-promoted polymerase chain reaction with the
first primer 5¢-CgaattcTAATACGACTCACTATAGGG
GGTGAAACGGTCTCG-3¢, containing the sense strand
sequence of the tmRNA 5¢-end and the T7 promoter, and
the second primer 5¢-CGTGAATTCATGCATGGTGGA
GGTGGGGGGAG-3¢, containing the antisense strand
sequence of the tmRNA-3¢-end and a NsiI restriction site
(underlined). The obtained DNA fragment without any
additional treatment was ligated to the linear pCR2.1 vector
for TA cloning (Invitrogen). E. coli B843 (DE3) cells
transformed with the resulting plasmid were plated onto
Luria–Bertani plates with 75 lgÆmL
)1
ampicillin and grown
for 10 h at 37 °C. All colonies contained the plasmid with
the ssrA-insert. The nucleotide sequence of the isolated
recombinant plasmids was checked by the dideoxy method
on both strands. In the obtained constructs, the ssrA-insert
was found in two different orientations in relation to the
body of the pCR2.1 vector. The variant designated pCR2.1-
A1L3 (Fig. 1) was selected for further studies.
Synthesis and purification of the tmRNA transcript
The pCR2.1-A1L3 plasmid was isolated from 10 g of
transformed E. coli cells. Prior to use, the plasmid was
treatedwiththeNsiI restriction enzyme, so that the 423 bp

CTP, 30 m
M
GMP, 80 mgÆmL
)1
PEG 8000,
75–100 lgÆmL
)1
DNA template and 100 lgÆmL
)1
T7 RNA
polymerase. After 6 h of incubation the reaction mixture
Fig. 1. Construction of the pCR2.1-A1L3 plasmid carrying the
T. thermophilus tmRNA-encoding sequence (striped arrow) under the T7
promoter. Orientation of the T7 promoters is shown by triangles.
464 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003
was phenol extracted and RNA was purified by HPLC on a
mixed-mode ionic-hydrophobic sorbent, methyltrioctylam-
ine-coated LiChrosorb RP-18 matix [16], followed by
preparative gel-electrophoresis in 7% polyacrylamide gel
with 7.8
M
urea. Usually the tmRNA transcript was
annealed prior to use by quick heating to 80 °Cin50m
M
Hepes/NaOH (pH 7.6), 1 m
M
MgCl
2
, followed by slow
cooling down to 20 °C.

impregnated with trichloroacetic acid. Then the filters were
extensively washed with ice-cold 5% trichloroacetic acid to
remove free amino acid. Trichloroacetic acid-insoluble
radioactivity was measured by liquid scintillation counting.
Structural analysis tmRNA melting curves were recorded
in a Varian Cary 50 spectrophotometer equipped with a
thermocontrolled cuvette holder. Measurements were per-
formed in 50 m
M
Hepes/NaOH (pH 7.6), 1 m
M
MgCl
2
,
0.1 m
M
Na
2
-EDTA. The temperature was increased at a
rate of 0.34 °CÆmin
)1
in the range 18–90 °C. The experiment
was performed in duplicate.
Ala$tmRNA and Ala$tRNA deacylation protection
assays
The protective effect of EF-Tu against spontaneous hydro-
lysis of the Ala$tmRNA or Ala$tRNA ester bond was
studied upon quick dissolution of the dry pellet of purified
[
3

H]Ala$tmRNA or 16 n
M
[
3
H]Ala$tRNA, 2.0 m
M
GDPNP, 90 m
M
Hepes/NaOH (pH 7.6), 10 m
M
MgCl
2
,
10 m
M
NH
4
Cl, 0.3 m
M
Spermine, 0.5 m
M
dithiothreitol,
0.25 m
M
Na
2
-EDTA. The time course of the
[
3
H]Ala$tmRNA and [

contained 3–12 l
M
EF-Tu*GDPNP, 1.0 D
260
units per mL
of uncharged tmRNA transcript, 1.5–4.5 m
M
GDPNP,
100 m
M
Hepes/NaOH (pH 7.6), 10 m
M
MgCl
2
,10m
M
NH
4
Cl, 0.3 m
M
Spermine, 0.5 m
M
dithiothreitol, 0.25 m
M
Na
2
-EDTA, 10% (v/v) glycerol. After 10 min of incubation
at 30 °C the solution was kept on ice for another 10 min
and then subjected to electrophoresis in nondenaturing 6%
polyacrylamide gel, with 25 m

Ala$tmRNA and Ala$tRNA hydrolytic decay was per-
formed with the use of the ÔLSW Data Analysis ToolboxÕ
add-in (MDL Information Systems, Inc) for Microsoft
EXCEL
.
Results
The sequence of the ssrA gene of T. thermophilus HB8
determined in this study differs in a single base (G
310
instead
of A
310
) from the previously reported complete ssrA
sequences of T. thermophilus strains HB8 [22] and HB27
(database of T. thermophilus HB27 genomic sequences at
Go
¨
ttingen Genomics Laboratory website, http://
www.g2l.bio.uni-goettingen.de). Guanine in position 310
was also found by Martindale and Williams in a partial
sequence of the ssrA gene from strain HB8 (T. thermophilus
tmRNA sequence, version 2, deposited 04/11/2000 at The
tmRNA website, This
minor difference can possibly be explained by an intraspe-
cific genomic variation. The presumed secondary structure
of T. thermophilus tmRNA resembles that of E. coli
tmRNA (Fig. 2).
T. thermophilus tmRNA was synthesized by run-off
transcription with the ssrA gene under the T7 promoter as
Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 465

84.3% of the total number of base pairs (in the case of
E. coli tmRNA this parameter amounts to only 57.5%).
Therefore, it was natural to expect a high resistance of
T. thermophilus tmRNA to thermoinduced unfolding.
Indeed, tmRNA melting experiments revealed no structural
changes in the temperature range from 18 °Cto70°C.
Noticeable transitions were registered only above 73 °C
(Fig. 4). Under similar conditions (1 m
M
MgCl
2
,near-
neutral pH), the melting profiles of E. coli tmRNA exhi-
bited two peaks, around 25 °Cand57°C, and the interval
of structural constancy was only from 30 °Cto45 °C[23]or
even more narrow [5]. Remarkably, even in the absence of
any stabilizing protein cofactors the unmodified T. thermo-
philus tmRNA transcript can sustain heating up to the
temperatures compatible with the efficient growth of this
thermophilic bacterium.
The apparent initial rate of the tmRNA aminoacylation
with T. thermophilus alanyl-tRNA synthetase was found to
be maximal at 50 °C. A similar activity profile was observed
inthecaseoftRNA
Ala
charging (Fig. 5). This is somewhat
lower than the optimal temperature of tRNA aminoacyla-
tion reported for cloned T. thermophilus alanyl-tRNA
synthetase (% 60 °C) [13]. Other Thermus synthetases
exhibit maximal activity at even higher temperatures:

explained considering that the monitored accumulation of
charged RNA in solution is determined by the balance
between enzyme-catalysed aminoacylation of RNA and
spontaneous deacylation of aminoacyl-RNA. Studies on
aminoacyl-tRNA stability revealed the alanyl ester bond to
be one of the most susceptible to hydrolytic cleavage.
Therefore, alanyl-tRNA synthetase encounters more intense
deacylation of charged RNA than synthetases of other
specificities. As a result, the measured maximum of the
apparent initial rate of RNA alanylation is shifted towards
lower temperatures and may float depending on the
concentration of alanyl-tRNA synthetase in the reaction
mixtures and on its specific activity in different buffers.
In order to characterize the substrate properties of the
tmRNA transcript, we attempted to estimate the kinetic
parameters of the tmRNA alanylation. A standard
approach based on the Michaelis–Menten scheme of the
enzyme-catalysed reaction was considered inadequate at the
conditions of our experiments. The corresponding constants
k
cat
and K
m
are usually calculated from the dependence of
Fig. 3. Analysis of the synthetic transcript of T. thermophilus tmRNA.
(A) Non-denaturing 2% agarose gel stained with ethidium bromide.
0.004 D
260
units (lane 1) and 0.001 D
260

Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 467
the initial reaction rates on the substrate concentration.
However, at elevated temperatures fast spontaneous
Ala$tmRNA hydrolysis disguised the real velocity of
tmRNA charging and shortened the linear part of amino-
acylation kinetics to the level, where correct measurement of
the initial reaction rate was barely possible. Another serious
problem was associated with the uncertainty of the molar
concentration of chargeable transcript in the reaction
mixtures. The extent of tmRNA aminoacylation was
varying dramatically depending on the reaction conditions
(temperature, buffer composition, enzyme concentration),
the maximal observed level being about 45 pmol Ala/D
260
unit of tmRNA transcript. Therefore, the estimates of the
total tmRNA concentration based on the quantification of
[
3
H]Ala coupled with tmRNA at the reaction plateau were
regarded as unreliable.
To circumvent these difficulties, we determined the
kinetic parameters of tmRNA aminoacylation by numerical
analysis of a set of reaction curves obtained at different
enzyme concentrations. A simplified scheme of the amino-
acylation mechanism included the reversible reaction of
Ala$tmRNA synthesis accompanied with the spontaneous
Ala$tmRNA hydrolysis:
E þ S
ÀÀ*
)ÀÀ

.
d½E=dt ¼Àk
f
½E½Sþk
b
½ESþk
cat
½ESÀk
rev
½E½P
ð3Þ
d½S=dt ¼Àk
f
½E½Sþk
b
½ESþk
h
½Pð4Þ
d½ES=dt ¼ k
f
½E½SÀk
b
½ESÀk
cat
½ESþk
rev
½E½Pð5Þ
d½P=dt ¼ k
cat
½ESÀk

ches its plateau very quickly, and the raising part of the
reaction curve is too short to be monitored accurately by the
filter technique. Therefore, we measured the kinetics of
tmRNA aminoacylation at an ATP concentration lowered
to 20 l
M
. By that way the specific activity of alanyl-tRNA
synthetase was decreased to the appropriate level, so that we
could use the desirable high enzyme-to-substrate ratios. The
proposed reaction mechanism was fitted to the experimental
dataset (five kinetic curves with 12 points each measured at
50 °C) with the use of the
DYNAFIT
program. In a control
experiment, T. thermophilus tRNA
Ala
was charged with
alanine under the same conditions, and the kinetic param-
eters of the reaction were determined by the same procedure
as in the case of tmRNA (Fig. 7, Table 2). The obtained
results reveal 5.9 times lower catalytic efficiency (k
cat
/K
m
)of
alanyl-tRNA synthetase with tmRNA as a substrate than
with tRNA
Ala
. The observed difference in substrate pro-
perties of tmRNA and tRNA

M
tRNA
Ala
). If the same proportion is
preserved at higher temperatures, the catalytic constant k
cat
for tRNA and tmRNA alanylation under standard reaction
conditions and 50 °C should be close to 0.8 s
)1
and 0.03 s
)1
,
respectively. This is to be compared with k
cat
values of
0.93 s
)1
[27], 1.1 s
)1
and 1.4 s
)1
[28] determined for E. coli
alanyl-tRNA synthetase and different isoacceptors of
Fig. 6. Kinetics of tmRNA aminoacylation with [
3
H]alanine by
T. thermophilus alanyl-tRNA synthetase upon temperature alterations.
A standard aminoacylation reaction mixture with 5 D
260
units per mL

protection upon formation of the canonical ternary complex
between EF-Tu, nucleotide cofactor and aminoacyl-tRNA.
While the velocity of Ala$tRNA
Ala
decay decreased
monotonously with the increase of EF-Tu*GDPNP con-
centration in the reaction mixture, the apparent rate of
Ala$tmRNA hydrolysis first decreased to a certain level
and then started to increase again (Fig. 9). The simplest
kinetic model that can describe this phenomenon implies
an existence of two interacting binding sites for
EF-Tu*GDPNP on tmRNA:
E þ P
ÀÀ*
)ÀÀ
k
1
k
À1
AP ð7Þ
E þ P
ÀÀ*
)ÀÀ
k
2
k
À2
BP ð8Þ
E þ AP
ÀÀ*

50 °C. The aminoacylation reaction mixtures contained all compo-
nents at standard concentrations except ATP whose concentration was
decreased to 20 l
M
. The drawing represents an output of the
DYNAFIT
program, where lines correspond to the best fit of the experimental
points to the proposed reaction mechanism. (A) Concentration of
alanyl-tRNA synthetase was 0.30 (circles), 0.60 (squares), 1.50 (trian-
gles), 3.00 (reverse triangles) and 4.50 (diamonds) l
M
. (B) Concen-
tration of alanyl-tRNA synthetase was 0.034 (circles), 0.068 (squares),
0.102 (triangles), 0.171 (reverse triangles), 0.342 (diamonds) l
M
.
Table 2. Kinetic parametes of tmRNA and tRNA
Ala
aminoacylation
with T. thermophilus alanyl-tRNA synthetase at 50 °C.
tmRNA tRNA
Ala
Constants Value
Standard
error Value
Standard
error
k
f
,m

b
/k
f
, l
M
0.354 1.319
K
m
¼ (k
b
+ k
cat
)/k
f
, l
M
0.361 1.432
k
cat
/K
m
,
M
)1
s
)1
265 1560
k
h
,10

interaction can be represented by a nonlinear system of
differential equations:
d½E=dt ¼Àk
1
½E½Pþk
À1
½APÀk
2
½E½Pþk
À2
½BP
À k
3
½E½APþk
À3
½ABPÀk
4
½E½BP
þ k
À4
½ABPð13Þ
d½P=dt ¼Àk
1
½E½Pþk
À1
½APÀk
2
½E½Pþk
À2
½BPÀk

4
½E½BP
À k
À4
½ABPð17Þ
Fig. 8. Kinetics of aminoacylation of the tmRNA transcript with alanyl-
tRNA synthetase in presence (black diamonds) or in absence (grey
squares) of Th. aquaticus EF-Tu*GDPNP. A45-lL aliquot with 23 l
M
EF-Tu*GDPNP complex in the exchange buffer (25 m
M
Hepes/
NaOH (pH 7.9), 5 m
M
GDPNP, 0.2
M
NH
4
Cl, 2 m
M
b-mercapto-
ethanol) was added to 150 lL of the standard aminoacylation reaction
mixture 20 s before the aminoacylation was started. In the case of the
control reaction mixture, 45 lL of the exchange buffer was added to
150 lL of the standard reaction mixture.
Fig. 9. Dependence of the apparent velocity of Ala$tmRNA (A) and
Ala$tRNA
Ala
(B) hydrolysis on the concentration of the EF-
Tu*GDPNP complex in the deacylation reaction mixture. The drawing

on the fitting efficiency was determinative. In general, when
k
1
, k
3
and k
h
were fixed, the fitting quality could not be
significantly improved by compensatory adjustment of all
the remaining parameters. The second group contained
parameters k
-2
, k
-3
, k
4
, which could vary 5–6 orders of
magnitude without serious effect on the deviation of the
model from the experimental data. The third group included
parameters k
-1
, k
2
, k
-4
, whose variability upon fitting was
more moderate than in the previous case and depended on
the current values of k
1
, k

, k
3
and k
h
) have been fixed. Then the
limits of admissible dispersion for each rate constant of the
third group were studied by systematic sampling of the (k
-1
,
k
2
, k
-4
)-space, while other kinetic parameters were kept fixed
at their currently best values. The kinetic constants were
characterized either by an optimized value within a 95%
confidence interval, or by the upper or lower limit that was
defined as the point where the stable increase of the standard
deviation reaches 1% of its minimal value at the current
conditions. Then the rate contstants of the second group
were estimated in the same way. The full set of the rate
constants was then refined by repeating an optimization
procedure, which assumed an improvement of the fitting
quality through the adjustment of the kinetic parameters
belonging to one group, while the rate constants from the
two other groups remained fixed. After two cycles of this
refinement, further adjustment of the kinetic parameters
could not decrease the difference between the model and the
experimental data anymore.
For comparison, the kinetic parameters of Ala$tRNA

). Therefore, the first
event in a major sequence of elementary interactions of
EF-Tu with Ala$tmRNA should be the formation of a
complex between EF-Tu*GDPNP and the alanylated
acceptor stem of tmRNA, in which the aminoacyl residue
is protected against hydrolysis. Then, the second EF-Tu
molecule binds to the alternative site on tmRNA. This
causes a quick ejection of the first EF-Tu molecule from the
canonical binding site, which is expressed by the drastic
increase of the corresponding dissociation rate constant (k
-4
is approximately 5 orders of magnitude higher than k
-1
). As
a result, the alanyl ester bond loses the protection and
becomes susceptible again to the nucleophilic attack of
hydroxyl anions (Fig. 10).
To test experimentally our suggestion that Ala$tmRNA
possesses a second EF-Tu binding site besides its alanylated
tRNA-like module, we checked whether EF-Tu*GDPNP
can form a complex with uncharged tmRNA. By analogy
with tRNA, we assumed that efficient EF-Tu binding to the
tRNA-like module of tmRNA is only possible when
tmRNA is aminoacylated. EF-Tu*GDPNP and tmRNA
were mixed and incubated for 10 min at the same conditions
as those used in the studies on Ala$tmRNA protection with
EF-Tu. Electrophoretic separation of these mixtures
revealed a change of tmRNA mobility in the presence of
the elongation factor (Fig. 11). Thus, even being uncharged,
tmRNA still retains an ability to bind EF-Tu*GDPNP.

2
, l
M
)1
s
)1
0.0805 0.0251
k
-2
,s
)1
0.01
a
upper limit
k
3
, l
M
)1
s
)1
0.149 0.006
k
-3
,s
)1
10
a
upper limit
k

-3
being equal approximately to 1000 k
-2
.
b
The
value has been evaluated from the kinetics of alanyl ester bond
hydrolysis in the absence of EF-Tu, and was fixed upon fitting the
model to the main massif of the experimental data.
Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 471
alanyl-tRNA synthetase and elongation factor Tu. Despite
the lack of post-transcriptional modifications, the tmRNA
transcript possessed a remarkable thermostability, which
may to a certain extent be explained by the large number of
GC base pairs in double-stranded regions. tmRNA melting
profile indicated structural constancy of this molecule in the
temperature range 18–70 °C. This made us sure that the
conformational state of the tmRNA transcript remains
essentially the same under different thermal conditions of
activity assays. The observed structural constancy makes
T. thermophilus tmRNA a good target for structural
studies.
The thermal optima of tmRNA and tRNA
Ala
amino-
acylation with T. thermophilus alanyl-tRNA synthetase
were found to be lower than the optimum of tRNA
Ala
charging reported by Lechler et al.[13](% 50 °Cvs.
% 60 °C, respectively). However, the real discrepancy may

or Ala$tmRNA at 70–
80 °Ctooccurin vivo. Among those could be the protection
Fig. 10. Proposed mechanism of EF–Tu*GDPNP interaction with
Ala$tmRNA. The size of the arrows reflects the relative magnitude of
the corresponding first-order or pseudo first-order kinetic constants at
micromolar concentrations of EF-Tu.
Fig. 11. Gel mobility shift study of the interaction between EF-Tu*GDPNP and uncharged tmRNA. Two equivalent gels were run at identical
conditions and stained with Coomassie Blue R250 (A) and with pyronin Y enhanced by silver treatment (B). Mixtures containing 1.0 D
260
units per
mL of tmRNA transcript and 0 (lanes 2), 3.8 (lanes 3), 7.5 (lanes 4) and 11.3 l
M
(lanes 5) EF-Tu*GDPNP in 80 m
M
Hepes/NaOH (pH 7.6), 8 m
M
MgCl
2
,0.5m
M
Spermine*4HCl, 10% (v/v) glycerol were incubated for 10 min at 30 °C, then for 10 min in ice-cold water bath, and then separated
in nondenaturing 8% polyacrylamide gel at room temperature, 120 V, for 2.5 h. The separation pattern of the mixture containing EF-Tu*GDPNP
alone is shown on lanes 1. Lanes 6 and 7 represent two different amounts of EF-Tu*GDP loaded.
472 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003
of the unstable aminoacyl ester bond in a complex with
elongation factor Tu, increase of the intracellular concen-
tration of alanyl-tRNA synthetase, or improvement of the
substrate properties of tRNA
Ala
and tmRNA in presence of

numerical method requires preliminary simulation studies
on the selected kinetic scheme in order to get a represen-
tation on how the experiment should better be organized to
provide reliable estimates of the desirable parameters. Also,
the use of a nonstandard kinetic model of the enzymatic
reaction may hamper the direct comparison of the results
with those obtained by other research groups. Under these
circumstances, adequate control experiments are absolutely
necessary.
The kinetic parameters of the alanyl-tRNA synthetase-
catalysed aminoacylation of the tmRNA transcript were
compared with those of tRNA
Ala
. The calculated values of
the association rate constant, k
f
, were found to be similar for
both RNA substrates. At the same time, the dissociation rate
of the tmRNA transcript from the complex with the enzyme
(k
b
) was 5.7 times slower than that of tRNA
Ala
. It could be
interpreted in the way that initial binding of alanyl-tRNA
synthetase to both tRNA
Ala
and tmRNA proceeds through
the interaction with similarstructural patterns (the alanylated
acceptor stem with the specifically recognizable GU base

correlates with that between the
corresponding k
f
values and allow us to suggest a similar
RNA recognition mechanisms for both the forward and
reverse reaction.
The alanyl residue is transferred onto the 3¢-end of the
enzyme-bound tmRNA transcript 23 times slower com-
pared with tRNA
Ala
. Such a difference may reflect non-
optimal positioning of the tmRNA CCA-terminus in the
active site of the enzyme. The 3¢-and5¢-heterogeneities of
the tmRNA transcript may also contribute to this effect,
because they do not prevent its binding to alanyl-tRNA
synthetase but impair its aminoacylation. Because of the
exceptionally low k
cat
value, the substrate properties of
tmRNA in the aminoacylation reaction expressed by the
k
cat
/K
m
ratio are noticeably worse than those of tRNA
Ala
.
Still, the difference is not as overwhelming as that between
E. coli tRNA
Ala

for the T. thermophilus tmRNA on the basis of the empirical
rule for tRNA molar UV-absorbance calculation [29].
Taking into account that the charging capacity of the
tmRNA transcript is 426 pmol per D
260
unit, we can
conclude that the extent of tmRNA aminoacylation in our
experiments did not exceed 10%. This is in agreement with
the observations of the research groups working with E. coli
tmRNA transcripts. In the absence of protein cofactors (like
EF-Tu or SmpB) no more than half of the total population
of E. coli tmRNA molecules could be charged [5,7,23,30,31].
The minimal model that efficiently describes the interac-
tion between thermophilic EF-Tu and tmRNA assumes the
presence of two interacting EF-Tu binding sites on
Ala$tmRNA. One of those corresponds to the tRNA-like
module of tmRNA and was designated as the canonical
EF-Tu binding site. EF-Tu*GDPNP affinity towards the
canonical site can be characterized by an equilibrium
dissociation constant of 0.058 l
M
, which is close to the K
d
values of the canonical ternary complexes between EF-Tu,
GTP and aminoacyl-tRNA. The location of another EF-Tu
binding site designated as the alternative one is unknown.
The alternative site reveals itself through the influence on
protein binding to the canonical site. When the alternative
site is occupied by EF–Tu, the interaction of either alanyl-
tRNA synthetase or EF-Tu with the tRNA-like module of

AAG
165
, which is identical to the nucleotides 2677 through
2684 of T. thermophilus 23S rRNA. These nucleotides are
located in the a-sarcin loop that is considered to interact
with EF-Tu upon binding of the ternary complex to the
ribosome. A similar sequence, ACCGAAG, was found in a
family of RNA aptamers selected for tight binding to
T. thermophilus EF-Tu in both the GTP and GDP form
[32]. Values of the equilibrium dissociation constants for
complexes between T. thermophilus EF-Tu, GTP (GDP)
and the RNA aptamers, which supposedly resemble the
a-sarcin loop of 23 rRNA (K
d
’s 10
)7
)10
)8
M
), converge
with our estimate of the K
d
for EF-Tu*GDPNP bound in
the alternative site of T. thermophilus tmRNA
(K
d
< 0.125 l
M
). Thus, if EF-Tu interacts with the alter-
native binding site on T. thermophilus tmRNA in the same

vity of tmRNA could be modulated by EF-Tu binding to
the alternative site. However, it seems unlikely that EF-Tu is
the only factor, which regulates in vivo the Ôon/offÕ state of
tmRNA, otherwise tmRNA would be permanently inactive
because of the high intracellular concentration of this
protein. The EF-Tu-promoted shutdown of tmRNA may
be counteracted by other components of the trans-transla-
tion pathway.
An interesting parallel to the observed interaction
between bacterial EF-Tu and Ala$tmRNA is presented
by wheat EF-1a binding to the 3¢ untranslated region of
tobacco mosaic virus genomic RNA [33]. Two different
binding sites for the elongation factor in this part of the viral
RNA have been found. One of them corresponds to the
tRNA-like structure at the very 3¢-end of the genomic RNA,
and interacts with EF-1a*GTP after being charged with
histidine. Another specific EF-1a binding site is located
within the upstream pseudoknot domain, and in that case
EF-1a binding does not depend on aminoacylation of the
viral RNA. The authors have suggested that the interaction
of EF-1a with the second site may contribute to the
regulation of the viral RNA translation on the host
ribosomes. Similarly, the binding of thermophilic EF-Tu
to the alternative site on tmRNA may affect the translation
efficiency of the tag-encoding tmRNA fragment.
The mechanism of interaction between the canonical and
alternative EF-Tu binding sites can be only guessed at. It
seems doubtful that simple sterical hindrance between
bound EF-Tu molecules is the only cause of the observed
negative cooperativity upon EF-Tu binding to the T. ther-

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