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 d
Trang 1tmRNA 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 70C The thermal optimum of tmRNA
alanylation was determined to be around 50C At 50 C,
tmRNA transcript was aminoacylated by alanyl-tRNA
synthetase with 5.9times lower efficiency (kcat/Km)
than tRNAAla, primarily because of the difference in
turnover numbers (kcat) Studies on EF-Tu protection of AlatmRNA 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; elevated temperatures; 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 tRNAAla,
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 tRNAAla 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: jnb@imsb.au.dk
Abbreviations: AlaRSase, alanyl-tRNA synthetase; GDPNP,
guano-sine 5¢-(b,c-imidotriphosphate) or 5¢-guanylylimidodiphosphate.
Enzymes: alanyl-tRNA synthetase, EC 6.1.1.7; EF-Tu, (EC 3.6.1.48).
(Received 4 October 2002, revised 18 November 2002,
accepted 27 November 2002)
Eur J Biochem 270, 463–475 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03401.x
Trang 2mixture 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 AlatmRNA The balance between these
two processes changes when the catalytic efficiency of
tmRNA alanylation (kcat/Km) becomes higher under the
influence of SmpB, or when synthesized AlatmRNA 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 AlatmRNA
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 AlatmRNA A
hot environment may in this way imprint the character
of the specific interactions between the macromolecules
involved in trans-translation in T thermophilus Here we
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-[2,3–3H]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 tRNAAla
with an amino-acid acceptance of 860 pmol per D260unit
was purified by successive chromatographies on Sepharose
4B, BND-cellulose and DEAE-Sephadex A-50 columns
Alanyl-tRNA synthetase from T thermophilus HB8 (Mw
195 kDa) with a specific activity of 105 nmolÆmin)1Æmg)1
(40C) was obtained generally according to Lechler et al
[13] T aquaticus EF-Tu (Mw45 kDa) was overproduced in
Escherichia coliSCS1 carrying plasmid pTacTU2 with the
tufA gene and purified as described in [14] T7 RNA
polymerase was overproduced in E coli BL21 carrying
plasmid pAR1219and 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)1ampicillin and grown for 10 h at 37C 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 treated with the NsiI 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 37C in a reaction mixture containing 40 mMTris/HCl (pH 8.0), 26 mMMgCl2, 5 mM
dithiothreitol, 0.5 mM Spermine, 0.5 mM Spermidine, 0.01% (v/v) Triton X100, 4 mM ATP, 4 mM UTP, 8 mM
GTP, 8 mM CTP, 30 mM GMP, 80 mgÆmL)1PEG 8000, 75–100 lgÆmL)1DNA template and 100 lgÆmL)1T7 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.
Trang 3was 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 7.8M urea Usually the tmRNA transcript was
annealed prior to use by quick heating to 80C in 50 mM
Hepes/NaOH (pH 7.6), 1 mM MgCl2, followed by slow
cooling down to 20C
Aminoacylation assays
Unless otherwise mentioned, the aminoacylation reaction
mixture contained 2.5 mM ATP, 12 mM MgCl2, 50 mM
Hepes/NaOH (pH 7.6 at 20C), 15 lM L-[3H]alanine,
0.5 mM Spermine, 0.05–2 lM 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 theL-[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 mM Hepes/NaOH (pH 7.6), 1 mM MgCl2,
0.1 mM Na2-EDTA The temperature was increased at a
rate of 0.34CÆmin)1in the range 18–90C The experiment
was performed in duplicate
AlatmRNA and AlatRNA deacylation protection
assays
The protective effect of EF-Tu against spontaneous
hydro-lysis of the AlatmRNA or AlatRNA ester bond was
studied upon quick dissolution of the dry pellet of purified
[3H]AlatmRNA or [3H]AlatRNA 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] [3H]AlatmRNA 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 mM sodium acetate (pH 5.0), and from
uncharged tmRNA by chromatography on acetylated
DBAE–cellulose (Serva) at 4C [17] Alanylated tmRNA
was collected into a tube with ice-cold 0.5Msodium 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 lMEF-Tu*GDPNP,
35 nM[3H]AlatmRNA or 16 nM[3H]AlatRNA, 2.0 mM
GDPNP, 90 mM Hepes/NaOH (pH 7.6), 10 mM MgCl2,
10 mM NH4Cl, 0.3 mM Spermine, 0.5 mM dithiothreitol,
0.25 mM Na2-EDTA The time course of the
[3H]AlatmRNA and [3H]AlatRNA 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, [3H]AlatmRNA and [3H]AlatRNAAla decay could be described by pseudo-first order kinetics characterized by the corresponding apparent rate constant kappand by the initial deacylation rate kapp[AlatmRNA]t¼ 0or kapp[AlatRNAAla]t¼ 0 Gel mobility shift assays
The standard mixture for mobility shift assays (10 lL) contained 3–12 lMEF-Tu*GDPNP, 1.0 D260units per mL
of uncharged tmRNA transcript, 1.5–4.5 mM GDPNP,
100 mM Hepes/NaOH (pH 7.6), 10 mM MgCl2, 10 mM
NH4Cl, 0.3 mMSpermine, 0.5 mMdithiothreitol, 0.25 mM
Na2-EDTA, 10% (v/v) glycerol After 10 min of incubation
at 30C the solution was kept on ice for another 10 min and then subjected to electrophoresis in nondenaturing 6% polyacrylamide gel, with 25 mM Tris-Borate (pH 8.3), 1.0 mM 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 reacsimula-tion networks were performed with the use of theDYNAFITprogram generally according to theDYNAFITReference 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, kapp values, from the kinetics of the AlatmRNA and AlatRNA hydrolytic decay was per-formed with the use of the LSW Data Analysis Toolbox 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 (G310instead
of A310) 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, http://www.indiana.edu/tmrna) 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
FEBS 2003 tmRNA from Thermus thermophilus (Eur J Biochem 270) 465
Trang 4a 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 mMMgCl2and 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
maritimaand 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 18C to 70 C
Noticeable transitions were registered only above 73C
(Fig 4) Under similar conditions (1 mM MgCl2, near-neutral pH), the melting profiles of E coli tmRNA exhi-bited two peaks, around 25C and 57 C, and the interval
of structural constancy was only from 30C to 45 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 50C A similar activity profile was observed
in the case of tRNAAlacharging (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 65C [24], isoleucyl-tRNA synthetase at 70C [25], phenylalanyl-tRNA synthetase at
70C [25] or 78 C [26] Therefore we checked whether the decline of the tmRNA alanylation rate above 50C is caused by irreversible degradation of any of the components
of the aminoacylation reaction mixture tmRNA was charged at 30C until a stable plateau was reached, then the tube with the reaction mixture was incubated at 80C for 20 min and transferred back to 30C The time course
of the AlatmRNA synthesis upon these temperature alterations is shown on Fig 6 Heating the reaction mixture
to 80C resulted in quick decrease of the AlatmRNA concentration to almost zero level However when it was cooled back to 30C, 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.
Trang 5ingredients 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
kcatand Kmare 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.
FEBS 2003 tmRNA from Thermus thermophilus (Eur J Biochem 270) 467
Trang 6the initial reaction rates on the substrate concentration.
However, at elevated temperatures fast spontaneous
AlatmRNA 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/D260
unit of tmRNA transcript Therefore, the estimates of the
total tmRNA concentration based on the quantification of
[3H]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
AlatmRNA synthesis accompanied with the spontaneous
AlatmRNA hydrolysis:
Eþ S *)kf
kb ES *)kcat
P!kb
where E, S and P represent alanyl-tRNA synthetase,
tmRNA and AlatmRNA, respectively, and ES is a
transient complex between the enzyme and tmRNA The corresponding system of differential Eqns (3–6) contained five adjustable parameters, kf, kb, kcat, krevand kh
ð3Þ
Additionally, the total concentration of tmRNA (designa-ted S0), 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 Kdand
Km, the enzyme concentration should be varied in the same interval where S0, Kdor Kmare 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 lM 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
50C) with the use of theDYNAFITprogram In a control experiment, T thermophilus tRNAAla 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 5.9times lower catalytic efficiency (kcat/Km) of alanyl-tRNA synthetase with tmRNA as a substrate than with tRNAAla The observed difference in substrate pro-perties of tmRNA and tRNAAlashould be attributed to a significantly lower kcatin 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*tRNAAlacomplex
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 mMto 20 lMin the reaction mixture results in a 345-fold drop of the specific activity of the enzyme at 40C (from 105 nmolÆmin)1Æmg)1 to 0.304 nmolÆmin)1Æmg)1 in the presence of 4 lMtRNAAla) If the same proportion is preserved at higher temperatures, the catalytic constant kcat for tRNA and tmRNA alanylation under standard reaction conditions and 50C should be close to 0.8 s)1and 0.03 s)1, respectively This is to be compared with kcat values of 0.93 s)1[27], 1.1 s)1and 1.4 s)1[28] determined for E coli alanyl-tRNA synthetase and different isoacceptors of
Fig 6 Kinetics of tmRNA aminoacylation with [3H]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 C to 80 C and backward during
measure-ments of the amount of [3H]AlatmRNA synthesized.
Trang 7E colitRNAAlaat 37C, or with the kcatvalue of 0.71 s)1
calculated from the specific activity of T thermophilus
alanyl-tRNA synthetase at 60C with unfractionated
tRNA as a substrate [13]
In order to characterize the interaction of EF-Tu with AlatmRNA, we attempted to study EF-Tu protection of AlatmRNA against spontaneous base-promoted hydro-lysis By analogy with E coli tmRNA [5], we expected to observe an increase of AlatmRNA yield in the amino-acylation reaction and a decrease of AlatmRNA 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 AlatmRNA 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 AlatRNAAla decay decreased monotonously with the increase of EF-Tu*GDPNP con-centration in the reaction mixture, the apparent rate of AlatmRNA 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:
Eþ P *)k1
Eþ P *)k2
k 2
Eþ AP *)k3
k 3
Eþ BP *)k4
k 4
P!kb
where E, P and S correspond to EF-Tu*GDPNP, AlatmRNA and deacylated tmRNA, respectively, AP represents a complex in which EF-Tu is bound to the acceptor stem of AlatmRNA 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 tRNAAlaaminoacylation with T thermophilus alanyl-tRNA synthetase at 50 °C.
tmRNA tRNAAla
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)3s)1 4.57 ± 2.12 26.1 ± 7.9
k cat , 10)3s)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)3s)1 2.21 ± 0.192.59± 0.16
S 0 , l M 1.59± 0.11 0.125 Number of datapoints 60 59
FEBS 2003 tmRNA from Thermus thermophilus (Eur J Biochem 270) 469
Trang 8ternary 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 AlatmRNA 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 AlatmRNA/EF–Tu
interaction can be represented by a nonlinear system of
differential equations:
ð14Þ
ð15Þ
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 A 45-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 AlatmRNA (A) and AlatRNA 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.
Trang 9d h h ð18Þ
The experimental dataset contained the values of the initial
AlatmRNA deacylation rates determined at seven
differ-ent concdiffer-entrations 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
AlatmRNA 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 k1, k3and kh, whose influence
on the fitting efficiency was determinative In general, when
k1, k3 and khwere 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, k4, 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, k2, k-4, whose variability upon fitting was
more moderate than in the previous case and depended on
the current values of k1, k3and kh
At the first stage, the value of khwas estimated from the
kinetics of AlatmRNA decay in the absence of EF-Tu
The search for k1 and k3 was performed by systematic
sampling of the (k1, k3)-space when all the remaining
constants (except kh) were allowed to be adjusted in order to
reach minimal standard deviation of the model from the
experimental data for each given pair of k1, k3 At the point
where the fitting quality was maximal, all the parameters of
the first group (k1, k3 and kh) 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,
k2, 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 AlatRNAAla
protection by EF-Tu*GDPNP were calculated using the
same approach The AlatRNAAladecay in the presence of
the elongation factor was described by the Eqns (7) and (11),
where P and S represented alanylated and deacylated
tRNAAla, 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 AlatRNAAlawhen 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 Kdvalue being increased at least 105times
On the other hand, the first EF-Tu molecule should bind to AlatmRNA predominantly at the canonical site, because the corresponding association rate (k1) is 13 times higher than that for the alternative site (k2) Therefore, the first event in a major sequence of elementary interactions of EF-Tu with AlatmRNA 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 AlatmRNA 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 AlatmRNA 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 AlatmRNA and AlatRNA Ala
pro-tection against alkaline hydrolysis at 40 °C by Th aquaticus EF-Tu in complex with GDPNP.
Constants
AlatmRNA AlatRNA 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 0.1490.006
k -3 , s)1 10 a upper limit
k 4 , l M )1 s)1 0.9upper limit
k -4 , s)1 5000 lower limit
k h , 10)3s)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 bThe 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.
FEBS 2003 tmRNA from Thermus thermophilus (Eur J Biochem 270) 471
Trang 10alanyl-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 18–70C 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 tRNAAla amino-acylation with T thermophilus alanyl-tRNA synthetase were found to be lower than the optimum of tRNAAla charging reported by Lechler et al [13] ( 50 C vs
60 C, respectively) However, the real discrepancy may
be smaller, taking into account that in both studies the initial aminoacylation velocity was measured in 10C steps, 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
80C was observed, in contrast to the above-cited paper where irreversible thermoinduced precipitation of the enzyme from 65C 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-lusgrowth (Topt72–75C, Tmax85C) This may be due to the intense spontaneous deacylation of AlatRNAAlaor AlatmRNA, 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 AlatRNAAlaor AlatmRNA at 70–
80C to occur in vivo Among those could be the protection
Fig 10 Proposed mechanism of EF–Tu*GDPNP interaction with
AlatmRNA 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 , 0.5 m 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.