Báo cáo khoa học: tmRNA from Thermus thermophilus Interaction with alanyl-tRNA synthetase and elongation factor Tu pptx

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.

This molecule possesses both mRNA and tRNA activities
and contains easily recognizable mRNA-like and tRNA-
like modules [1]. The latter is formed by converging-3¢-and
5¢-termini of the 300–400 nucleotide-long chain. The main
biological function of tmRNA is to relieve ribosomes that
remain for a long time in complex with mRNA without
elongating the polypeptide chain. Such a situation arises
upon translation of truncated mRNA deprived of stop-
codon, or intact mRNA with clustered rare codons. The
intervention of tmRNA may also take place in the case
when the ribosomes idle at the mRNA stop-codon awaiting
proper termination of translation [2].
As a first step of the tmRNA-assisted ribosome rescue
(called trans-translation), the aminoacylated tRNA-like
module of the tmRNA binds to the A-site of stalled 70S
ribosomes with peptidyl-tRNA in the P-site. The polypep-
tide chain is transferred onto the 3¢-end of the tmRNA in
the course of the transpeptidation reaction. Then the tRNA-
like module, now carrying the polypeptide, moves into
the ribosomal P-site. At the same time, the first codon of the
mRNA-like part of the tmRNA enters the A-site and the
reprogrammed ribosome resumes the polypeptide chain
elongation by adding approximately 10 aminoacyl residues
to the synthesized protein. When the stop-codon of the
mRNA-like module is reached, the translation is terminated
in the usual way [3]. Thus, the trans-translation results both
in release of the arrested ribosome and in labelling the newly
formed protein with a standard C-terminal peptide tag that
serves as a signal for degradation by specific proteases.
During its functioning, tmRNA interacts with a number

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

cofactors.
Aminoacylation of tmRNA is an absolute prerequisite of
its activity in trans-translation [11]. However, tmRNA
charging with alanyl-tRNA synthetase in vitro in the
absence of other proteins was found to be slow and
inefficient in comparison with tRNA alanylation. SmpB
improves significantly the substrate properties of tmRNA
and induces a rise of the plateau of the tmRNA alanylation
reaction [5,8]. An addition of EF-Tu*GTP to the reaction
Correspondence to J. Nyborg, Institute of Molecular and Structural
Biology, University of Aarhus, Gustav Wieds Vej 10C,
DK-8000 Aarhus C, Denmark.
Fax: + 45 8612 3178, Tel.: + 45 8942 5257,
E-mail:
Abbreviations: AlaRSase, alanyl-tRNA synthetase; GDPNP, guano-
sine 5¢-(b,c-imidotriphosphate) or 5¢-guanylylimidodiphosphate.
Enzymes:alanyl-tRNAsynthetase,EC6.1.1.7;EF-Tu,(EC3.6.1.48).
(Received 4 October 2002, revised 18 November 2002,
accepted 27 November 2002)
Eur. J. Biochem. 270, 463–475 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03401.x
mixture was reported to increase further both the rate and
the yield of tmRNA alanylation [5]. The observed stimu-
lative effects of SmpB and EF-Tu on tmRNA charging were
interpreted in terms of the dynamic interplay of synthetase-
catalysed aminoacylation of tmRNA and spontaneous
deacylation of Ala$tmRNA. The balance between these
two processes changes when the catalytic efficiency of
tmRNA alanylation (k
cat
/K

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

H]Alanine (42.0 CiÆmmol
)1
) was from Amersham
Life Science, GDPNP (5¢-guanylylimidodiphosphate),
GMP, GTP, ATP, UTP, CTP, Spermidine*3HCl and
Spermine*4HCl were products of Sigma, all other chemicals
were from Fluka and AppliChem. T. thermophilus tRNA
Ala
with an amino-acid acceptance of 860 pmol per D
260
unit
was purified by successive chromatographies on Sepharose
4B, BND-cellulose and DEAE-Sephadex A-50 columns.
Alanyl-tRNA synthetase from T. thermophilus HB8 (M
w
195 kDa) with a specific activity of 105 nmolÆmin
)1
Æmg
)1
(40 °C) was obtained generally according to Lechler et al.
[13]. T. aquaticus EF-Tu (M
w
45 kDa) was overproduced in
Escherichia coli SCS1 carrying plasmid pTacTU2 with the
tufA gene and purified as described in [14]. T7 RNA
polymerase was overproduced in E. coli BL21 carrying
plasmid pAR1219 and purified as described in [15]. Calf
liver alkaline phosphatase immobilized on agarose beads
was from Sigma. All restriction enzymes, Taq DNA
polymerase and T4 DNA ligase were from New England

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

DNA fragment containing the tmRNA-encoding sequence
under the T7 promoter was cut out of the pCR2.1-A1L3
construct. The 3¢-overhangs of the obtained DNA duplexes
were removed by treatment with E. coli exonuclease I. The
423-bp DNA fragment was separated from the rest of the
plasmid by size-exclusion chromatography on Sephacryl S-
500 H (Pharmacia) and used as a template for T7 RNA
polymerase-catalysed run-off transcription. The tmRNA
synthesis was performed at 37 °C in a reaction mixture
containing 40 m
M
Tris/HCl (pH 8.0), 26 m
M
MgCl
2
,5m
M
dithiothreitol, 0.5 m
M
Spermine, 0.5 m
M
Spermidine,
0.01% (v/v) Triton X100, 4 m
M
ATP, 4 m
M
UTP, 8 m
M
GTP, 8 m
M

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.

Aminoacylation assays
Unless otherwise mentioned, the aminoacylation reaction
mixture contained 2.5 m
M
ATP, 12 m
M
MgCl
2
,50m
M
Hepes/NaOH (pH 7.6 at 20 °C), 15 l
ML
-[
3
H]alanine,
0.5 m
M
Spermine, 0.05–2 l
M
chargeable RNA and 1–
10 lg/mL of alanyl-tRNA synthetase (referred to as
standard aminoacylation conditions). The velocity of the
aminoacylation was measured by the rate of the
L
-[
3
H]ala-
nine covalent attachment to RNA. At appropriate times
aliquots were taken out of the reaction mixture by a lambda
pipette and spotted onto Whatman 3MM paper filters

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$tmRNA or [
3
H]Ala$tRNA in a EF-Tu*GDPNP-
containing mixture, preincubated for 10 min at the appro-
priate temperature. The EF-Tu*GDPNP complex was
prepared as described in [14]. [
3
H]Ala$tmRNA was
synthesized under standard conditions, treated with phenol,
separated from low-molecular-mass components of the
reaction mixture by gel-filtration on Sephadex G-25 (Phar-
macia) in 50 m
M
sodium acetate (pH 5.0), and from
uncharged tmRNA by chromatography on acetylated
DBAE–cellulose (Serva) at 4 °C [17]. Alanylated tmRNA
was collected into a tube with ice-cold 0.5
M
sodium acetate
(pH 5.0), quantified by radioactivity, divided into appro-
priate portions and precipitated with 3 vol. of ethanol. The
pellet was dried using SpeedVac. Alanylated tRNA was
passed through the same procedure. The deacylation
reaction mixtures contained 0.38–4.52 l
M
EF-Tu*GDPNP,
35 n
M
[
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 [

3
H]Ala$tRNA hydrolysis was
monitored by the filter technique. All kinetics of the
decay reaction were characterized by 11 datapoint each.
Lambda pipettes were used to take out aliquots from the
reaction mixtures. In all cases, [
3
H]Ala$tmRNA and
[
3
H]Ala$tRNA
Ala
decay could be described by pseudo-
first order kinetics characterized by the corresponding
apparent rate constant k
app
and by the initial deacylation
rate k
app
[Ala$tmRNA]
t ¼ 0
or k
app
[Ala$tRNA
Ala
]
t ¼ 0
.
Gel mobility shift assays
The standard mixture for mobility shift assays (10 lL)

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

M
Tris-Borate (pH 8.3),
1.0 m
M
magnesium acetate as gel and running buffer, at
room temperature and 12 VÆcm
)1
for 2.5 h. The experi-
ments were performed in duplicate for separate RNA and
protein visualization. In order to visualize RNA only, the
gels were stained with pyronin Y [18] with consecutive silver
enhancement according to Blum et al. [19]. The location of
EF-Tu bands on the gels was revealed by staining with
Coomassie Brilliant Blue R-250.
Mathematical treatment of the kinetic data
General numerical analysis of the kinetic data and simula-
tion studies on the model reaction networks were performed
with the use of the
DYNAFIT
program generally according to
the
DYNAFIT
Reference Manual and [20,21]. Unless other-
wise mentioned, the desirable kinetic parameters were
determined within a 95% confidence interval by a least-
squares regression procedure based on the Levenberg–
Marquardt fitting algorithm. Evaluation of the apparent
rate constants, k
app
values, from the kinetics of the

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

a template. The transcript was purified by HPLC on a
mixed-mode ionic-hydrophobic matrix followed by prepar-
ative urea-PAGE. The obtained RNA was annealed in
presence of 1 m
M
MgCl
2
and analysed by gel-electrophor-
esis (Fig. 3). The tmRNA transcript migrated as a single
band during separation under denaturing conditions. At the
same time, nondenaturing gel-electrophoresis in agarose
revealed few faint satellite bands following the main one.
The major RNA species was isolated from the agarose gel
and reannealed. However, when it was subjected again to
the electrophoretic separation under identical conditions,
the presence of the same high-molecular-mass admixtures
was observed. Such a heterogeneity of the tmRNA
transcript is thus likely to be caused by the reversible
RNA oligomerization. As a macromolecule with numerous
self-complementary stretches, tmRNA may be prone to
form intermolecular contacts instead of the equivalent
intramolecular ones. Taking into consideration that the
presumed oligomers account for a relatively small fraction
of the transcript population, we used the obtained RNA
without further purification.
The distinctive feature of the T. thermophilus tmRNA is
its anomalously high GC content even in comparison with
tmRNAs from the more extreme thermophiles, Thermotoga
maritima and Aquifex aeolicus (Table 1). The percentage of
GC pairs in predicted double-stranded regions is equal to

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:

glutamyl-tRNA synthetase at 65 °C [24], isoleucyl-tRNA
synthetase at 70 °C [25], phenylalanyl-tRNA synthetase at
70 °C[25]or78°C [26]. Therefore we checked whether the
decline of the tmRNA alanylation rate above 50 °Cis
caused by irreversible degradation of any of the components
of the aminoacylation reaction mixture. tmRNA was
charged at 30 °C until a stable plateau was reached, then
the tube with the reaction mixture was incubated at 80 °C
for 20 min and transferred back to 30 °C.Thetimecourse
of the Ala$tmRNA synthesis upon these temperature
alterations is shown on Fig. 6. Heating the reaction mixture
to 80 °C resulted in quick decrease of the Ala$tmRNA
concentration to almost zero level. However when it was
cooled back to 30 °C, recharging of tmRNA occured with
almost the same rate and to the same extent as it was before
the thermal jump. This indicates that all the initial
Fig. 2. Secondary structure of Thermus
thermophilus tmRNA. Four pseudoknots are
labelled pK1, pK2, pK3 and pK4. Trinucleo-
tides that encode amino acids of the tag-
peptide are boxed. Helix numbering is given
according to Zwieb et al. (1999) [34]. The
ambiguous nucleotide at position 310 is
marked by the arrow.
466 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003
ingredients of the aminoacylation reaction mixture remain
undamaged upon prolonged incubation at the highest
temperature used in our study.
The decreased thermal optimum of the alanyl-tRNA
synthetase activity observed in our experiments may be

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

units (lane 3) of the tmRNA
transcript were separated on the gel in the presence of DNA markers
[lane 2, 100 bp DNA ladder (New England Biolabs)]. (B) Denaturing
8% polyacrylamide gel stained with pyronin Y. Lanes 1 and 2 show
separation patterns of the samples equivalent to 0.1 and 0.01 lL,
respectively, of the standard transcription reaction mixture after the
tmRNA synthesis was completed.
Table 1. Correlation between growth temperature and tmRNA
GC-content for selected bacterial species.
Bacterial species
Growth
temperature, °C
tmRNA
total GC
content (%)
Optimum Maximum
Aquifex aeolicus 85 95 66.8
Thermotoga maritima 80 90 62.1
Thermus thermophilus 72 85 70.5
Bacillus stearothermophilus 60 75 59.6
Escherichia coli 37 45 52.9
Fig. 4. UV-absorbance melting curve of the purified T. thermophilus
tmRNA transcript.
Fig. 5. Temperature dependence of apparent initial rate of aminoacy-
lation of the T. thermophilus tmRNA transcript (black circles) and
tRNA
Ala
(grey squares) by the homologous alanyl-tRNA synthetase. The
dependence is expressed as the relative aminoacylation activity, with
100% corresponding to the maximal observed initial reaction rate.

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

k
f
k
b
ES
ÀÀ*
)ÀÀ
k
cat
k
rev
E þ P ð1Þ
P À!
k
b
S ð2Þ
where E, S and P represent alanyl-tRNA synthetase,
tmRNA and Ala$tmRNA, respectively, and ES is a
transient complex between the enzyme and tmRNA. The
corresponding system of differential Eqns (3–6) contained
five adjustable parameters, k
f
, k
b
, k
cat
, k
rev
and k
h

.
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

rev
½E½PÀk
h
½Pð6Þ
Additionally, the total concentration of tmRNA (designa-
ted S
0
), which was the same in all the reaction mixtures, had
to be searched for. Preliminary simulation studies on the
above-mentioned kinetic model revealed some constraints
on the possible organization of the kinetic experiment. The
most important limitation was that in order to obtain
maximally reliable estimates of the total concentration of
functional tmRNA and of the affinity parameters K
d
and
K
m
, the enzyme concentration should be varied in the same
interval where S
0
, K
d
or K
m
are expected to be found (i.e. in
the micromolar range). Also, the kinetic curve should be
well sampled on different stages of the reaction progress.
However, at comparable concentrations of alanyl-tRNA
synthetase and tmRNA, the aminoacylation reaction rea-

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

Ala
should be attributed to a
significantly lower k
cat
in the case of tmRNA alanylation.
At the same time, alanyl-tRNA synthetase possesses slightly
higher affinity towards tmRNA, mostly because of slower
dissociation of the AlaRSase*tmRNA complex in compar-
ison with the AlaRSase*tRNA
Ala
complex.
With certain caution we can extrapolate some of our
results to standard aminoacylation conditions, taking into
account the fact that the decrease of ATP concentration
from 2.5 m
M
to 20 l
M
in the reaction mixture results in a
345-fold drop of the specific activity of the enzyme at 40 °C
(from 105 nmolÆmin
)1
Æmg
)1
to 0.304 nmolÆmin
)1
Æmg
)1
in
thepresenceof4l

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

of the purified tmRNA transcript and 5 n
M
of alanyl-tRNA synthetase
was transferred from 30 °Cto80°C and backward during measure-
ments of the amount of [
3
H]Ala$tmRNA synthesized.
468 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003
E. coli tRNA
Ala
at 37 °C, or with the k
cat
value of 0.71 s
)1
calculated from the specific activity of T. thermophilus
alanyl-tRNA synthetase at 60 °C with unfractionated
tRNA as a substrate [13].
In order to characterize the interaction of EF-Tu with
Ala$tmRNA, we attempted to study EF-Tu protection of
Ala$tmRNA against spontaneous base-promoted hydro-
lysis. By analogy with E. coli tmRNA [5], we expected to
observe an increase of Ala$tmRNA yield in the amino-
acylation reaction and a decrease of Ala$tmRNA deacy-
lation rate in the nonenzymatic hydrolytic reaction in the
presence of EF-Tu and 5¢-Guanylylimidodiphosphate
(GDPNP), a stable analog of GTP. Surprisingly, tmRNA
charging with alanine was found to be strongly inhibited by
the elongation factor (Fig. 8). Moreover, the influence of
EF-Tu on the Ala$tmRNA deacylation rate revealed a
deviation from the mechanism of aminoacyl ester bond

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*

)ÀÀ
k
3
k
À3
ABP ð9Þ
E þ BP
ÀÀ*
)ÀÀ
k
4
k
À4
ABP ð10Þ
P À!
k
b
S ð11Þ
BP À!
k
b
BS ð12Þ
where E, P and S correspond to EF-Tu*GDPNP,
Ala$tmRNA and deacylated tmRNA, respectively, AP
represents a complex in which EF-Tu is bound to the
acceptor stem of Ala$tmRNA in the same way as in the
Fig. 7. Kinetics of aminoacylation of the tmRNA transcript (A) and
tRNA
Ala
(B) at different concentrations of alanyl-tRNA synthetase at

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

M
)1
s
)1
12.9 ± 3.8 19.8 ± 7.3
k
b
,10
)3
s
)1
4.57 ± 2.12 26.1 ± 7.9
k
cat
,10
)3
s
)1
0.0956 ± 0.0079 2.23 ± 0.56
k
rev
,m
M
)1
s
)1
0.313 ± 0.081 0.874 ± 0.182
K
d
¼ k

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

)3
s
)1
2.21 ± 0.19 2.59 ± 0.16
S
0
, l
M
1.59 ± 0.11 0.125
Number of datapoints 60 59
Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 469
ternary complexes between EF-Tu, GTP and aminoacyl-
tRNAs (therefore the alanylated tmRNA acceptor stem is
further referred to as the canonical EF-Tu binding site), BP
represents a complex in which EF-Tu is bound to a
hypothetical alternative site, and ABP represents a complex
in which EF-Tu molecules are bound simultaneously to
both the canonical and alternative sites. Qualitatively, the
observed dependence of the Ala$tmRNA deacylation rate
on EF-Tu concentration may result from negative cooper-
ativity upon EF-Tu binding to the canonical and alternative
sites, if we assume that (a) the protein protects the
aminoacyl ester bond only when it is bound to the canonical
site, and (b) the canonical site has higher affinity towards
EF-Tu*GDPNP than the alternative one.
In order to quantify the interrelationships between the
elementary reactions of the proposed kinetic model, we
attempted to determine the corresponding kinetic constants
or, at least, to estimate the limits of their admissible
dispersion. The dynamics of the Ala$tmRNA/EF–Tu

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

h
½P
ð14Þ
d½AP=dt ¼ k
1
½E½PÀk
À1
½APÀk
3
½E½APþk
À3
½ABP
ð15Þ
d½BP=dt ¼ k
2
½E½PÀk
À2
½BPÀk
4
½E½BPþk
À4
½ABP
À k
h
½BPð16Þ
d½ABP=dt ¼ k
3
½E½APÀk
À3
½ABPþk

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

represents an output of the
DYNAFIT
program, where lines correspond
to the best fit of the experimental points to the proposed reaction
mechanism.
470 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003
d½S=dt ¼ k
h
½Pþk
h
½BPð18Þ
The experimental dataset contained the values of the initial
Ala$tmRNA deacylation rates determined at seven differ-
ent concentrations of EF-Tu*GDPNP in the reaction
mixture. The numerical analysis of the model was accom-
plished through an iterative procedure, which took advant-
age of the fact that the elementary reactions of the kinetic
model contribute differently to the initial rate of
Ala$tmRNA decay. Briefly, on the basis of preliminary
simulation tests of the model, the kinetic constants were
divided into three groups according to their influence on the
fitting quality characterized by the standard deviation of the
theoretical curve from the experimental data. The first
group consisted of parameters k
1
, k
3
and k
h
, whose influence

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

3
and k
h
.
At the first stage, the value of k
h
was estimated from the
kinetics of Ala$tmRNA decay in the absence of EF-Tu.
The search for k
1
and k
3
was performed by systematic
sampling of the (k
1
, k
3
)-spacewhenalltheremaining
constants (except k
h
)wereallowedtobeadjustedinorderto
reach minimal standard deviation of the model from the
experimental data for each given pair of k
1
, k
3
. At the point
where the fitting quality was maximal, all the parameters of
the first group (k
1

, 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

Ala
protection by EF-Tu*GDPNP were calculated using the
same approach. The Ala$tRNA
Ala
decay in the presence of
the elongation factor was described by the Eqns (7) and (11),
where P and S represented alanylated and deacylated
tRNA
Ala
, respectively. The calculated rate constants are
listed in Table 3. The affinity of the elongation factor
towards the alanylated tRNA-like module of tmRNA (the
canonical binding site) does not seem to be much different
from its affinity towards Ala$tRNA
Ala
when the alternative
EF-Tu binding site on tmRNA is empty. However, when it
is occupied, EF-Tu binding to the canonical site deteriorates
dramatically, the K
d
value being increased at least 10
5
times.
On the other hand, the first EF-Tu molecule should bind to
Ala$tmRNA predominantly at the canonical site, because
the corresponding association rate (k
1
) is 13 times higher
than that for the alternative site (k
2

). 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.

Discussion
In the present study we investigated the interaction of
the T. thermophilus tmRNA transcript with thermophilic
Table 3. Kinetic parameters for Ala$tmRNA and Ala$tRNA
Ala
pro-
tection against alkaline hydrolysis at 40 °CbyTh. aquaticus EF-Tu in
complex with GDPNP.
Constants
Ala$tmRNA Ala$tRNA
Ala
Value
Standard
error Value
Standard
error
k
1
, l
M
)1
s
)1
1.03 0.08 0.368 0.027
k
-1
,s
)1
0.0602 0.0371 0.010 upper limit
k

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

4
, l
M
)1
s
)1
0.9 upper limit
k
-4
,s
)1
5000 lower limit
k
h
,10
)3
s
)1
0.90
b
0.04 0.89
b
0.04
a
Throughout the fitting procedure, strong covariation of k
-2
and
k
-3
was observed, 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

be smaller, taking into account that in both studies the
initial aminoacylation velocity was measured in 10 °Csteps,
and could be ascribed to the different composition of the
reaction mixtures. Also, in our experiments no irreversible
denaturation of alanyl-tRNA synthetase (or any other
component of the aminoacylation reaction mixture) at
80 °C was observed, in contrast to the above-cited paper
where irreversible thermoinduced precipitation of the
enzyme from 65 °C and upward was described. This
apparent disagreement is presumably due to a substrate
protection effect, which may occur in our case because of
the alanyl-tRNA synthetase stabilization in the presence of
tmRNA, alanine and ATP.
It is noteworthy that the in vitro determined thermal
optimum of alanyl-tRNA synthetase activity is significantly
lower than the characteristic temperatures of T. thermophi-
lus growth (T
opt
72–75 °C, T
max
85 °C). This may be due to
the intense spontaneous deacylation of Ala$tRNA
Ala
or
Ala$tmRNA, which quickly becomes comparable with the
enzyme-promoted aminoacylation reaction upon increase
of temperature. The finding raises a question about the
possible compensatory mechanisms, which allow an effi-
cient production of Ala$tRNA
Ala

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

cofactors (proteins, polyamines, metal ions). Also, com-
partmentalization of the components of the translational
apparatus and acceleration of the consumption of alanyl-
ated RNAs by the ribosome may reduce the negative
consequences of Ala$tRNA
Ala
or Ala$tmRNA instability
at elevated temperatures [12].
In our studies of the kinetics of tmRNA and tRNA
alanylation, we took advantage of the numerical analysis of
the experimental data with the use of the
DYNAFIT
program.
Because of the technical difficulties we could not use the
traditional approach, which is based on the simplified
presentation of an enzyme-catalysed reaction as a two-step
process and supposes data analysis according to the
approximate analytic solution of the corresponding differ-
ential equation (Michaelis–Menten model, modified later by
Briggs and Haldane). The kinetic characteristics of the
reaction are evaluated in that case from the dependence of
the initial reaction rate on the concentration of substrate. In
contrast, the numerical method permits a quantitative
analysis of much more complicated reaction mechanisms,
when the analytical solution of the corresponding system of
differential equations is barely possible. Unlike the tradi-
tional approach, it does not limit measurements by the
initial stages of the reaction and allows to include into the
analysis datapoints from other parts of the reaction progress
curve. On the other hand, the successful application of the

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

pair, and the T loop). On the other hand, in an established
complex with the enzyme, tmRNA may form more contacts
(not necessary specific ones) with alanyl-tRNA synthetase
than tRNA
Ala
. This should result in higher activation energy
of the dissociation of the alanyl-tRNA synthetase*tmRNA
complex and, consequently, in a smaller k
b
.
It is interesting to compare k
f
and k
rev
values, which
characterize the enzyme binding to uncharged and alanyl-
ated RNA, respectively. At the given conditions, both
Ala$tmRNA and Ala$tRNA
Ala
associate with alanyl-
tRNA synthetase much slower, than uncharged tmRNA
and tRNA
Ala
. This reveals insignificant product inhibition
in the aminoacylation reaction catalysed by T. thermophilus
alanyl-tRNA synthetase. The difference between k
rev
values
for tmRNA and tRNA
Ala

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

and tmRNA transcripts determined by
Barends et al. [5]. They reported a k
cat
/K
m
value for
tmRNA 75 times lower than for tRNA
Ala
. The poor
substrate properties of the tmRNA transcript were attri-
buted mostly to a high K
m
upon its aminoacylation with
E. coli alanyl-tRNA synthetase, however, the experimental
data presented by the authors do not seem to be sufficient
for a reliable separate determination of the k
cat
and K
m
parameters.
Alanine acceptance of the tmRNA transcript was esti-
mated to be 426 pmol per D
260
unit. This value came from
the comparison of the calculated molar concentration of the
transcript in the reaction mixtures and the corresponding
tmRNA-associated UV-absorbtion at 260 nm. It is surpris-
ingly close to the value of 398 pmol per D
260
unit predicted

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

tmRNA is strongly deteriorated. The ability of deacylated
tmRNA to form a complex with EF-Tu*GDPNP provides
another evidence in favour of the existence on tmRNA of a
EF-Tu binding site distinct from the tRNA-like module.
Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 473
Our conclusions correlate with the observations of
Zvereva et al. [31] on the interaction of a E. coli tmRNA
transcript with wild-type E. coli EF-Tu in the GDP or GTP
form. Their crosslinking and footprinting experiments
revealed uncharged tmRNA to have a binding site, which
can accommodate EF-Tu*GTP or EF-Tu*GDP. Nucleo-
tides located in helix 2 and pseudoknot pK4 were found to
be in contact with the elongation factor upon complex
formation. The affinities of EF-Tu*GTP and EF-Tu*GDP
towards uncharged tmRNA were very similar, and notably
lower than those of EF-Tu*GTP towards aminoacyl-
tRNAs. The authors hypothesize that the elongation factor
binds to uncharged tmRNA at the
312
ACCGA
316
sequence
(helix 2), which is also present in the a-sarcinloopof23S
rRNA. At the same time, they notice that this region is not
conserved among tmRNAs, and therefore their suggestion
can be valid only in the case of E. coli tmRNA. In this
context it seems interesting that T. thermophilus tmRNA
contains a eight nucleotide-long segment,
158
ACCGG

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

way as with the a-sarcin loop of 23S rRNA, the fragment
158
ACCGGAAG
165
is a likely candidate as EF-Tu binding
platform. However, such an interaction could not have a
universal character because the ACCGGAAG sequence is
not well conserved in tmRNAs.
In contrast with the results of Zvereva et al. [31], Barends
et al. [5] reported no detectable binding of uncharged E. coli
tmRNA transcript to overexpressed His-tagged E. coli
EF-Tu, neither in GTP nor in GDP form. The efficiency
of Ala$tmRNA protection against RNase A hydrolysis
was observed to increase monotonously with the increase of
EF-Tu*GTP concentration. Kinetic data did not indicate
the existence of any other EF-Tu binding site on tmRNA
than the tRNA-like module. This could be related to the
presence of the His-tag at the C-terminus of the cloned
elongation factor. The His-tag may block EF-Tu binding to
the alternative site on tmRNA.
The role of the alternative EF-Tu binding site on tmRNA
is unclear. When it is occupied, T. thermophilus tmRNA is
practically inactivated. Both aminoacylation of tmRNA
and EF-Tu protection of the preformed aminoacyl ester
bond become inefficient. Consequently, at elevated temper-
atures and supramicromolar concentrations of EF-Tu*GTP
the population of Ala$ tmRNA would quickly vanish
because of the intense spontaneous hydrolysis of the
unprotected aminoacyl ester bonds and the lack of
compensatory reaminoacylation. Thus, the biological acti-

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-

mophilus tmRNA transcript. The strong asymmetry of the
mutual influence of the canonical and alternative sites can
best be explained by structural perturbations of the tRNA-
like module induced by EF-Tu binding at the alternative site.
However, to make this issue clear requires a separate study.
Finally, the conclusion can be made that the efficiency of
T. thermophilus tmRNA aminoacylation at elevated tem-
peratures is cramped by the intense spontaneous hydrolytic
decay of synthesized Ala$tmRNA. At the same time, the
elongation factor Tu by itself does not provide
Ala$tmRNA with sufficient protection against deacylation,
whereas it efficiently stabilizes Ala$tRNA
Ala
. The observed
activity of EF-Tu on tmRNA can be described as regulatory
rather than protective. The complex between Ala$tmRNA
and EF-Tu bound at the canonical site has a transient
character. In contrast, deacylated tmRNA with EF-Tu in
the alternative binding site represents a kinetically stable
state if the protein concentration is high enough. Clearly, the
complete system of factors that prepare T. thermophilus
tmRNA entrance into the trans-translation reaction should
necessarily include some additional components besides
alanyl-tRNA synthetase and elongation factor Tu.
Acknowledgements
This work has been supported by the Danish Natural Science Research
Council through its Programme for Biotechnological Research.
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