In this study, the sequence encoding the coiled-coil motif in EF-Ts was deleted from the genome in Escherichia coli by gene replacement.The growth rate of the resulting mutant strain was
Trang 1Functional effects of deleting the coiled-coil motif
Henrik Karring1, Asgeir Bjo¨rnsson2, Søren Thirup1, Brian F C Clark1and Charlotte R Knudsen1
1
Department of Molecular Biology, Aarhus University, Denmark;2deCODE Genetics, Inc., Reykjavik, Iceland
Elongation factor Ts (EF-Ts) is the guanine
nucleotide-exchange factor for elongation factor Tu (EF-Tu) that is
responsible for promoting the binding of
aminoacyl-tRNA to the mRNA-programmed ribosome.The
struc-ture of the Escherichia coli EF-Tu–EF-Ts complex reveals
a protruding antiparallel coiled-coil motif in EF-Ts, which
is responsible for the dimerization of EF-Ts in the crystal
In this study, the sequence encoding the coiled-coil motif
in EF-Ts was deleted from the genome in Escherichia coli
by gene replacement.The growth rate of the resulting
mutant strain was 70–95% of that of the wild-type strain,
depending on the growth conditions used.The mutant
strain sensed amino acid starvation and synthesized the
nucleotides guanosine 5¢-diphosphate 3¢-diphosphate and
guanosine 5¢-triphosphate 3¢-diphosphate at a lower
cell density than the wild-type strain.Deletion of the coiled-coil motif only partially reduced the ability of EF-Ts to stimulate the guanine nucleotide exchange in EF-Tu.However, the concentration of guanine nucleo-tides (GDP and GTP) required to dissociate the mutant EF-Tu–EF-Ts complex was at least two orders of mag-nitude lower than that for the wild-type complex.The results show that the coiled-coil motif plays a significant role in the ability of EF-Ts to compete with guanine nucleotides for the binding to EF-Tu.The present results also indicate that the deletion alters the competition bet-ween EF-Ts and kirromycin for the binding to EF-Tu Keywords: elongation factor Ts; elongation factor Tu; guanine nucleotide exchange; kirromycin; (p)ppGpp
Elongation factor Tu (EF-Tu) and elongation factor Ts
(EF-Ts) are proteins known from the classical model of the
elongation cycle of protein synthesis in prokaryotes.EF-Tu,
which is a highly conserved G-protein, is active in the
GTP-bound form (EF-Tu–GTP) and inactive in the GDP-GTP-bound
form (EF-Tu–GDP).The equilibrium dissociation
con-stants for EF-Tu–GDP and EF-Tu–GTP are 1· 10)9and
5· 10)8M, respectively [1].The active EF-Tu–GTP binds
aminoacyl-tRNA (aa-tRNA) and promotes the binding of
the aa-tRNA to the A-site of the mRNA-programmed
ribosome.Upon codon recognition by a cognate ternary
complex (EF-Tu–GTP–aa-tRNA), the ribosomal GTPase
centre stimulates the GTPase activity of EF-Tu and the
bound GTP is hydrolysed.The inactive EF-Tu–GDP is
released from the ribosome and recycled to the active
EF-Tu–GTP by the exchange of GDP with GTP
[2].Stimula-tion of the guanine nucleotide release in EF-Tu by EF-Ts [3]
is required as the dissociation of GDP is otherwise very slow
(2· 10)3s)1) [1,4] I n vivo, the binding of GTP to the binary EF-Tu–EF-Ts complex is favoured owing to the ninefold higher concentration of GTP (0.9 mM) than GDP (0.1 mM) [5].The activation of EF-Tu is completed by the dissociation
of EF-Ts from EF-Tu–GTP.The equilibrium governing EF-Tu is further driven to the GTP-bound state by the formation of EF-Tu–GTP–aa-tRNA.Previous studies have indicated the existence of a structural isomerization in the EF-Tu–GDP–EF-Ts complex from a high- to a low-affinity nucleotide binding conformation [1,6,7].According to the results published by Gromadski et al.[1], the structures
of the binary EF-Tu–EF-Ts complex and the nucleotide-bound ternary complexes are different
EF-Ts in Escherichia coli is encoded by a single gene (tsf) located in the rpsB-tsf operon of the chromosome.The elongation factor consists of 282 residues and has a molecular mass of 30.3 kDa [8] The structure of the E coli EF-Tu–EF-Ts complex (Fig.1) reveals that EF-Ts is an elongated molecule containing four domains: the N-ter-minal domain; the core domain; the dimerization domain; and the C-terminal module [9].The dimerization domain (residues 180–228), which consists of a-helices 9, 10 and 11,
is inserted in subdomain C of the core domain and contains the protruding antiparallel coiled-coil motif (helices 10 and
11, residues 187–203 and 208–226) responsible for the dimerization of EF-Ts in the crystal.In the crystal of E coli EF-Tu–EF-Ts, a quaternary complex, formed by two molecules of each of the elongation factors, is observed The coiled-coil motifs of each of the two EF-Ts molecules form strong intimate contacts with each other, and therefore the tetramer is best designated as [EF-Ts]2)2EF-Tu However, the stoichiometry of the E coli EF-Tu–EF-Ts
Correspondence to C.R.Knudsen, Department of Molecular Biology,
Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C,
Denmark.Fax: + 45 8612 3178, Tel.: + 45 8942 5036,
E-mail: crk@imsb.au.dk
Abbreviations: aa-tRNA, aminoacyl-tRNA; CBD, chitin binding
domain; EF-G, elongation factor G; EF-Ts, elongation factor
Ts; EF-Ts mt , mitochondrial EF-Ts; EF-Tu, elongation factor Tu;
LB, Luria–Bertani; MCS, multiple cloning site; ppGpp, guanosine
5¢-diphosphate 3¢-diphosphate; pppGpp, guanosine 5¢-triphosphate
3¢-diphosphate; (p)ppGpp, ppGpp and pppGpp.
(Received 10 June 2003, revised 26 August 2003,
accepted 8 September 2003)
Trang 2complex used in the classical model of the elongation cycle
has remained 1 : 1 because only the heterodimer has been
detected in solution [10–12].Thus, it is believed that
dimerization of EF-Ts, as observed in the crystal structure,
is not physiologically relevant
The cellular response to amino acid starvation causes an
accumulation of the unusual guanine nucleotides guanosine
5¢-diphosphate 3¢-diphosphate (ppGpp) and guanosine
5¢-triphosphate 3¢-diphosphate (pppGpp) (stringent
response) [13,14].These compounds are synthesized by the
relAgene product when activated by codon-specific A-site
binding of uncharged tRNA, reflecting the limitation of the
corresponding amino acid [15,16].The guanosine
5¢-diphos-phate 3¢-diphosphate and guanosine 5¢-triphosphate
3¢-diphosphate [(p)ppGpp] nucleotides mediate a reduction
in the elongation rate and influence the accuracy of
translation.Early results indicated that (p)ppGpp affect
translation by inhibition of elongation factors Tu, Ts and G
[17–19].However, more recent studies suggest that
(p)ppGpp have no direct effects on either translation
elongation or error rates, but rather effect translation
indirectly by inhibition of mRNA synthesis during amino
acid starvation [20–23]
Kirromycin is one in a family of antibiotics that inhibits
protein synthesis by binding EF-Tu–GTP and preventing
the release of EF-Tu–GDP from the ribosome [24].These
antibiotics bind at the interface between domain 1 (guanine
nucleotide-binding domain, G-domain) and domain 3 of
EF-Tu, and lock EF-Tu–GDP in the GTP-bound
confor-mation after hydrolysis of the GTP at the ribosome [25]
Kirromycin has been shown to stimulate the nucleotide exchange in EF-Tu by increasing the dissociation of GDP and binding of GTP [26].In addition, the binding of kirromycin and EF-Ts to EF-Tu are mutually exclusive [27] Alignment of several EF-Ts sequences from different prokaryotes, as well as different eukaryotic organelles, shows preservation of the coiled-coil region (residues 187–226 in
E coli EF-Ts) only in prokaryotes and chloroplasts.The motif contains a number of well-conserved residues, such
as Glu193, Lys206, Pro207, Lys213, Gly217 and Arg218 (E coli numbering) of which most are located at the surface-exposed side of helix 11.In mammalian mitochondrial EF-Ts (EF-Tsmt, human and bovine) the region encompas-sing the coiled-coil motif is completely absent [28,29].These observations suggest that the coiled-coil motif of EF-Ts has
an important, but as yet unknown, role in bacteria The role of the coiled-coil motif of EF-Ts in the guanine nucleotide-exchange reaction in EF-Tu was investigated
by studying the functional effects of deleting the motif in EF-Ts, both in vivo and in vitro.The motif was deleted
in endogenous EF-Ts in E coli strain UY211 by gene replacement, and the phenotype of the resulting mutant strain (named GRd.tsf) was characterized In addition, the activity of the coiled-coil deleted EF-Ts mutant in promoting guanine nucleotide exchange in EF-Tu, and the stability of the mutant EF-Tu–EF-Ts complex in the presence of guanine nucleotides and kirromycin, were examined in vitro
The present study shows that the coiled-coil motif in EF-Ts is not required for the guanine nucleotide-exchange
Fig 1 Structure of the Escherichia coli elongation factor Tu (EF-Tu)–elongation factor Ts (EF-Ts) heterodimer EF-Ts is drawn as ribbons, indicating the secondary structure, whereas EF-Tu is drawn as a C-alpha trace.(A) The position of EF-Tu domains with respect to the N-terminal and core domains of EF-Ts.The structure shows that EF-Ts interacts with domain 1 (G-domain) and domain 3 of EF-Tu.(B) The view in (A) is rotated 90 around a vertical axis to show the positions of the protruding coiled-coil motif (residues 187–226) and the C-terminal module.The terminal positions of the deletion of the coiled-coil motif and the insertion of the linker peptide, EPGGEA, are indicated by residues Asp184 and Glu225, which are shown in ball & stick representation.Coordinates were obtained from PDB entry [1EFU] and displayed using MOLMOL [64].
Trang 3reaction in EF-Tu.Instead, the motif plays a significant role
in the ability of EF-Ts to compete with guanine nucleotides
for the binding to EF-Tu.We propose that the coiled-coil
motif in bacterial EF-Ts is involved in a structural
isomerization step of the nucleotide-bound EF-Tu–EF-Ts
complex during the guanine nucleotide-exchange reaction
Materials and methods
Construction of gene replacement vector
pMAK705-d.tsf
A tsf mutant, lacking the sequence encoding the coiled-coil
motif, was constructed by site-directed mutagenesis and
inserted into the gene replacement vector, pMAK705 [30],
for deletion of the motif in endogenous EF-Ts of E coli.The
exact terminal positions of the deletion in E coli EF-Ts
(residues 185–224) were decided upon by combining
know-ledge about the structure of the coiled-coil motif (Fig.1) with
alignment analysis of E coli Ts and mammalian
EF-Tsmt [28].According to the structure of E coli EF-Ts,
deletion of this small, isolated domain should not have a
major effect on the folding and stability of the remaining part
of EF-Ts.However, the distance between EF-Ts residues
Asp184 and Glu225 in the EF-Tu–EF-Ts complex was
measured and found to be 12.2 A˚.Therefore, to bridge this
gap in the resulting mutant, the hexapeptide EPGGEA,
which is found at the corresponding position in mature
bovine EF-Tsmt(residues 232–237) [31] and human EF-Tsmt
(residues 243–248) [28], was inserted as a linker (Figs 1 and 2)
Chromosomal DNA from E coli strain UY211 (ara,
D(lac-pro), nalA, thi) [32] was isolated and used as a template
in two separate PCR reactions to prepare fragments
flanking the sequence encoding the coiled-coil motif in EF-Ts.The fragments cover 200 base pairs of the genomic sequences upstream and downstream of tsf, respectively (Fig.2), to ensure a satisfactory frequency of homologous recombination for the gene replacement.The upstream fragment was prepared using forward primer tsf-UPS (5¢-ATGCGGGATCCAAGCTTGAGCTTACATC AGTAAGTGACCGGGATGA-3¢) and reverse primer Ts185 (5¢-TAGCACCCCGGGTTCGTCTTCCGGTTTG ATGAATTCTGGCTTG-3¢).Primer tsf-UPS contains a BamHI restriction site (underlined).Primer Ts185 contains
a unique AvaI restriction site (underlined) and part of a sequence encoding the hexapeptide EPGGEA (bold text) The downstream fragment was prepared using forward primer Ts224 (5¢-TAGTTCCCCGGGGGTGAAGCTGA AGTTTCTCTGACCGGTCAGCCGTTC-3¢) and reverse primer tsf-DOWNS (5¢-AGTCAGGATCCGTCGACA GAGCTTCGCCACTCAACTTAAGCAGAA-3¢).Like primer Ts185, Ts224 contains a unique AvaI restriction site (underlined) and part of a sequence encoding the hexapep-tide EPGGEA (bold text).Primer tsf-DOWNS contains a BamHI restriction site (underlined).Both PCR products were digested with AvaI and ligated.The ligation was used directly as a template in a final PCR using primers tsf-UPS and tsf-DOWNS Taq polymerase was used in all PCR amplifications.The amplified fragment containing the mutant tsf gene (d.tsf) was digested with BamHI and inserted into pMAK705.The sequence of the insert in the resulting pMAK705-d.tsf plasmid was verified by DNA sequencing
Deletion of the coiled-coil motif in endogenous
E coli EF-Ts
E colistrain UY211 was chosen for the gene replacement because of its almost wild-type genotype.Therefore, any unintended phenotypic side-effects of deleting the coiled-coil motif should not occur.The strain was transformed with pMAK705-d.tsf by electroporation, and selection for co-integrates and screening for regeneration of free plasmid was basically performed as described by Hamilton et al [30].Plasmids from single colonies were identified, following digestion with AvaI, as containing either wild-type or mutant tsf.Colonies carrying wild-type tsf on the pMAK705 vector were cured from the plasmid by sequen-tial inoculation and growth overnight three times in Luria– Bertani (LB) medium, without chloramphenicol, at 44C The genomic deletion in colonies of the mutant strain (GRd.tsf) (Fig 2) was verified by DNA sequencing Measurement of growth rates
A broad range of growth rates was obtained by culturing cells at 37C in various traditional and modified media based on LB, glucose/M9 or glucose/Mops [33,34].The minimal media contained 0.40 mM proline and 1.0 mM
thiamine to take into account auxotrophies.The concen-tration of glucose in the glucose/M9 and glucose/Mops media was 0.4% (w/v) For nutritional enrichments, the minimal media were supplemented with 0–3% (w/v) Casamino acid (Difco) or 0–3 times the recommended concentrations of -amino acids and other nutrients, except
Fig 2 Schematic illustration of gene organizations in Escherichia coli
strains and the gene replacement plasmid pMAK705-d.tsf (A)
Elonga-tion factor Ts (EF-Ts) wild-type strain UY211.(B) Gene replacement
plasmid pMAK705-d.tsf, used for the deletion of the coiled-coil motif
(cc) of endogenous EF-Ts.The plasmid contains an 1148-bp insert in
which the region in E coli tsf (bp 556–675) encoding the coiled-coil
motif has been exchanged with a sequence encoding the hexapeptide
EPGGEA found at the corresponding position in mammalian
EF-Ts mt (C) The coiled-coil deleted EF-Ts mutant strain GRd.tsf.Genes:
rpsB, gene encoding ribosomal protein S2; tsf, gene encoding wild-type
EF-Ts; cc, coiled-coil encoding region; smbA, gene encoding SmbA;
d.tsf, gene encoding coiled-coil deleted EF-Ts mutant.Restriction site
B: BamHI.
Trang 4for proline and thiamine [34].LB medium was modified
by replacing bacto-tryptone with various amounts of
Casamino acid (1–3%).Overnight cultures were inoculated
into 40 mL of media to a final attenuance D600of 0.1 or
D420of 0.07 The D600culture was used to analyse growth
in media based on LB, while the D420 culture was used
to analyse growth in the supplemented minimal media
Growth was followed by measuring D-values every
10–20 min.Data obtained from the exponential growth
phase after at least one population doubling (Dt> 2· D0)
were used to determine the rate constant, l, according to the
exponential growth equation [Dt¼ D0· exp(l · t)] [35]
Only growth curves with a correlation coefficient of
R > 0.99 were accepted
Growth in the presence of kirromycin (Sigma) was
performed at 37C in microtitre trays.LB medium
(120 lL), containing kirromycin at the concentrations
indicated, was inoculated with an overnight culture to a
D595 of 0.07 During growth, the D595 was measured
every 20–30 min by the use of an ELISA reader.For each
concentration of kirromycin, three independent growth
curves were recorded
Amino acid starvation and measurement of (p)ppGpp
levels
Cells from overnight cultures were washed in glucose/Mops
and inoculated at a D420of 0.14 in 25 mL of glucose/Mops
that contained 15 lCiÆmL)1of [32P]phosphate and 40 lM
of proline, which represents 0.1· the recommended amino
acid concentration [34].Cells were grown at 37C and the
cell density was followed by measuring the D420.Cell samples
of 50 lL were withdrawn at appropriate time-points and
mixed with 50 lL of 2 0M formic acid.The labelled cell
samples were run on TLC sheets [poly(ethylenimine)] and
developed as described by Cashel et al.[36] with the only
exception that 10 lL of the cell-free supernatant was spotted
on the thin layer sheets.(p)ppGpp levels were determined by
phosphor-imaging.Every (p)ppGpp measurement was
nor-malized to the cell density by division with the D420of the
culture at the corresponding time-point.To correct for the
additional phosphate group in pppGpp compared with
ppGpp, the ppGpp levels were multiplied by a factor of 1.25,
in accordance with previous results [36].The level of pppGpp
in UY211 before starvation was given a value of 1.0 The
dependency of growth inhibition on proline concentration
was determined by growing cells in glucose/Mops containing
different concentrations (20–60 lM) of proline
Cloning of EF-Ts and construction of the EF-Ts mutant
Wild-type E coli EF-Ts was cloned into expression plasmid
pAB146 (constructed by A.Bjo¨rnsson, unpublished results),
which is a derivative of plasmid pET11d that contains the
EcoRI–PstI Intein chitin binding domain (CBD) fragment
of plasmid pCYB3 from the IMPACT System (New
England Biolabs) [37].In contrast to pCYB3, which
contains one SapI restriction site, the multiple cloning site
(MCS) of pAB146 contains two SapI restriction sites.The
SapI sites (underlined) in the MCS (AAGAAGG
AGCTCTTCCATGGAATTCCTCGAGGGCTCTTCC
TGC) of pAB146 are designed for cloning of genes so that
no additional amino acids are introduced into the final protein product
Plasmid pGEX-tsf [38] was used as template in a PCR The applied primers both contain a SapI restriction site.In addition to the SapI restriction sites located in the primers, the E coli tsf has an internal SapI restriction site.After digestion of the PCR product with SapI, the resulting two fragments were inserted into pAB146 in a three-fragment ligation.The cloning was verified by DNA sequencing.The resulting plasmid pAB146-tsf was used as a template in PCR amplifications for the construction of the coiled-coil deleted E coli EF-Ts gene (d.tsf).The mutagenesis was basically performed as described for the construction of pMAK705-d.tsf, except that primers UPS and tsf-DOWNS were replaced with the plasmid-specific forward primer pT711 (5¢-TAATACGACTCACTATAGGGGA ATTG-3¢) and reverse primer Int R (5¢-CCCATGACCT TATTACCAACCTC-3¢), respectively.The final amplified fragment was digested with XbaI and KpnI and inserted into pAB146.The unique XbaI and KpnI restriction sites are positioned immediately upstream and downstream of the MCS of pAB146, respectively.The mutagenesis resulting in the construct pAB146-d.tsf was verified by DNA sequen-cing.Cloned Pfu polymerase (New England Biolabs) was used in all PCR amplifications
Expression and purification ofE coli EF-Tu and EF-Ts Expression and purification of E coli EF-Tu using plasmid pGEXFXtufA was performed essentially as previously described [39].Strain B834(DE3) (Novagen) was trans-formed with pAB146-tsf and pAB146-d.tsf for the expres-sion and purification of wild-type and mutant EF-Ts, respectively.LB medium containing 100 mgÆL)1of ampi-cillin was inoculated with 1% of an overnight culture and incubated at 37C until a D600 of 0.7 was reached Expression was induced at 30C for 3 h by adding 0.5 mM of isopropyl thio-b-D-galactoside.Cells were har-vested by centrifugation and resuspended in Column Buffer (20 mM Tris/HCl, pH 8.0, 0.5M NaCl, 0.1 mM EDTA,
10 mMMgCl2, 1 0 gÆL)1Triton-X-100, 15 lMGDP, 10% glycerol) to a final density of 0.25 gÆml)1of cells.Clarified cell extract was prepared by passing the cell slurry twice through a French Press followed by centrifugation at
12 000 g for 20 min.The lysate was treated with DNase I and loaded onto a chitin column (New England Biolabs), equilibrated with Column Buffer, at 4C.The chitin column was thoroughly washed with Column Buffer and thereafter equilibrated with Precleavage Buffer (20 mMTris/ HCl, pH 8.0, 50 mM NaCl, 0.1 mM EDTA).Then, the column was incubated at 25C for 16–24 h after fast equilibration with Cleavage buffer (Precleavage buffer containing 60 mM dithiothreitol).Pure EF-Ts was eluted with Elution Buffer (20 mMTris/HCl, pH 8.0, 0.5MNaCl, 0.1 mM EDTA, 1.0 gÆL)1 Triton-X-100) at 4C and dialysed against 20 mM Tris/HCl, pH 7.2, 40 mM KCl,
1 mM MgCl2, 0 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol.The concentration of EF-Ts was determined by amino acid analysis [40]
The EF-Tu–EF-Ts complex was formed by mixing equal amounts of EF-Tu and EF-Ts followed by dialysis in Buffer
D (20 m Tris/HCl, pH 7.6, 50 m KCl, 5 m EDTA,
Trang 51 mMdithiothreitol).This dialysis leads to the dissociation
of Mg2+ and GDP.The EF-Tu–EF-Ts complex was
purified on a 5-mL HiTrap Q column (Pharmacia) using a
100-mL 100–375 mM KCl gradient in 20 mM Tris/HCl,
pH 7.6, 1 mMdithiothreitol.The design of fusion proteins
and the methods used for purification ensured that the
recombinant proteins have the same number of amino acids
as the native elongation factors
Activity assays
The concentration of EF-Tu active in binding guanine
nucleotides, and the activity of EF-Ts in promoting the
exchange of guanine nucleotide with E coli EF-Tu–GDP,
was determined essentially as described previously [41],
except that cellulose acetate filters (Gelman Sciences) were
used for filter-binding.EF-Tu was 50% active in guanine
nucleotide binding.Both wild-type and mutant EF-Ts were
estimated to be 100% active based on their ability to bind
EF-Tu, as judged by purification of the EF-Tu–EF-Ts
complex in excess of EF-Tu by anion-exchange
chromato-graphy, as described above.The ability of EF-Ts to
stimulate the exchange of EF-Tu-bound GDP with free
[3H]GDP was determined at 0C.The reaction mixtures
contained about 0.35 lM active EF-Tu, 2.5 lM [3H]GDP
( 1100 c.p.m per pmol) and different concentrations of
EF-Ts (0–1.0 nM), as indicated.The reactions were initiated
by adding [3H]GDP, and 100-lL samples were withdrawn
every minute and filtered immediately.The filters were
washed three times with 3 mL of cold Wash buffer (10 mM
Tris/HCl, pH 7.6, 10 mM NH4Cl, 10 mM MgCl2) and
dissolved in OptiPhase HiSafe 3 (Fisher Chemicals) before
counting in a scintillation counter.The slope (pmol
exchanged GDP min)1) from each exchange reaction was
plotted as function of the added amount of EF-Ts.The
slope of the GDP exchange in the absence of EF-Ts was
subtracted from each slope value.The exchange of
EF-Tu-bound GDP with free [3H]GTP was performed as described
for [3H]GDP, except that pyruvate kinase and
phos-phoenolpyruvate were included in the [3H]GTP stock, so
that the final concentrations in the assay were 16.5 lgÆmL)1
and 12 lM, respectively
The ability of EF-Ts to stimulate EF-Tu in translation
in vitrowas determined by poly(U)-directed polymerization
of phenylalanine in the polymix system, as described by
Ehrenberg et al.[42] and modified by Pedersen et al.[43]
Reaction mixtures were systematically titrated with E coli
wild-type EF-Ts, yeast [14C]Phe-tRNAPhe, and ribosomes,
to ensure that the nucleotide exchange limited the
transla-tion.For the final experiments, the ribosome mix
con-tained 2.0 lM70S ribosomes and 2.6 mgÆmL)1poly(U).The
factor mix contained 8.0 lM [14C]Phe-tRNAPhe(44 c.p.m
per pmol), 112 nMactive EF-G, 40 nMactive EF-Tu and
0–8.0 nMEF-Ts.The ribosome mix and the factor mix were
incubated separately at 37C for 10 min before the reaction
was initiated by mixing 25 lL of each.Every 5 min, an 8 lL
sample was withdrawn for measurement of phenylalanine
polymerization.Data were handled, as described above for
the nucleotide exchange reactions.The 70S ribosomes were
prepared from E coli MRE600 cells, essentially as described
by Spedding [44].Yeast tRNAPhewas aminoacylated and
extracted as described previously [43]
Zone-interference gel electrophoresis The stability of the E coli EF-Tu–EF-Ts in the presence of guanine nucleotides and kirromycin was studied using the method of vertical zone-interference gel electrophoresis [45] Agarose gels (1.5% and 2.0%), prepared in electrophoresis buffer (20 mM Tris acetate, pH 7.6, 3.5 mM magnesium acetate) were used in the experiments.Zone solutions containing guanine nucleotide and kirromycin, at the concentrations indicated, were prepared in electrophoresis buffer containing 5% (v/v) glycerol.Sample solutions were prepared in 10% (w/v) sucrose containing a trace of bromphenol blue.Zone solutions of 80 lL, and sample solutions of 5 lL, which contained 50 pmol of the EF-Tu– EF-Ts complex, were pipetted into slots.The electropho-resis was performed at 8C for 1 h at a constant voltage of
300 V, and the gel was stained as previously described [45]
Results
Deletion of the coiled-coil motif in endogenous EF-Ts The sequence encoding the coiled-coil motif in tsf was deleted by gene replacement in E coli strain UY211, using plasmid pMAK705-d.tsf (Fig 2) The frequency of co-integrates in the gene replacement procedure was in the range of 10)5to 10)4.After regenerating free plasmid in the cells, colonies with deletion of the genomic sequence encoding the coiled-coil motif of EF-Ts were identified among the colonies harbouring free plasmid containing wild-type tsf.The deletion into the chromosome was verified directly by isolation of genomic DNA followed by PCR amplification and sequencing of the inserted DNA.The morphology of the resulting mutant strain (GRd.tsf) was compared with that of UY211 using a light microscope (cells not shown).No obvious differences in the size and shape of cells of GRd.tsf and UY211 were observed Growth rates
Growth rate constants (h)1) were determined from growth
at 37C in a range of traditional and modified media based
on LB medium, glucose/M9 or glucose/Mops.In all of the media tested, the growth rate of GRd.tsf was lower than that of UY211 (Fig.3).The reduction in the growth rate of GRd.tsf compared with the growth rate of UY211 was more pronounced in media based on LB than in supple-mented minimal media.Therefore, the growth rates obtained in LB-based media could not be grouped with those obtained in supplemented minimal media.The maximal growth rate of UY211 was 1.6 and 1.85 h)1in the groups of supplemented minimal media and that of media based on LB, respectively.For strain GRd.tsf, the maximal growth rate was 1.3 h)1in both groups of media Therefore, the growth rate of GRd.tsf ranges from 70– 95% of that of UY211 (Fig.3).The lowest growth rate of GRd.tsf (70%), relative to the growth rate of UY211, was obtained in LB medium where both strains expressed their maximal growth rate.The absence of the coiled-coil motif does not affect growth at rates below 0.8 h)1.In contrast, the mutant strain seems to be impaired in media supporting higher growth rates.This disadvantage caused by deletion
Trang 6of the coiled-coil motif is dependent on growth rate, i.e the
higher the potential growth rate (above 0.8 h)1), the larger
the impairment
Phenotype during amino acid starvation
The phenotypes of UY211 and GRd.tsf during amino
acid starvation were determined by culturing cells at 37C
in glucose/Mops containing growth-limiting amounts of
proline (auxotrophic amino acid) (Fig.4) Instantaneous
growth inhibition caused by proline exhaustion was observed
in both cases.However, strain GRd.tsf was found to be more
sensitive to the depletion of proline than the wild-type strain
Proline exhaustion caused growth inhibition of GRd.tsf at a
significantly lower cell density compared with UY211
(Fig.4A).Thus, the point of growth inhibition of GRd.tsf
(D420¼ 0.47), represented by the D420value of the culture at
the interception point between the exponential and the
starvation phase of growth, is 78% of the point of growth inhibition of UY211 (D420¼ 0.60) The point of growth inhibition of GRd.tsf when expressed as a percentage of that
of UY211, was independent of the initial proline concentra-tion used (data not shown).In comparison, the mutant and the wild-type strains reached the same cell density in the stationary phase in both supplemented minimal media and in
LB (data not shown)
The synthesis of (p)ppGpp was measured (Fig.4B) to determine if the starvation sensitive phenotype of GRd.tsf was the result of abnormal levels of (p)ppGpp.During exponential growth, the (p)ppGpp basal levels were similar in both stains (UY211 D420< 0.575; GRd.tsf:
D420< 0.475) At the points of growth inhibition, ppGpp and pppGpp appear both in UY211 and GRd.tsf owing to the auxotrophic exhaustion of proline (Fig.4A,B).After rapid increases to maxima, the levels of (p)ppGpp decrease
to plateaus, which are maintained.No significant differences were observed between the levels and the production patterns of the (p)ppGpp in the two strains, either before
or during the full stringent response (Fig.4B)
Growth rates in the presence of kirromycin The effect of kirromycin on the growth of GRd.tsf was investigated by measuring growth rates of cultures in LB containing various concentrations of kirromycin.During growth in microtitre trays, where aeration is suboptimal, strains UY211 and GRd.tsf had similar growth rates ( 0.85 h)1) in the absence of kirromycin.The growth rates of both strains decreased as the concentration of kirromycin was increased from 0.5 to 5.0 lM.However, the reduction in the growth rate caused by the presence of kirromycin was larger for strain GRd.tsf than for the wild-type strain (Fig.5).Thus, at 50 lMkirromycin, the growth rate of GRd.tsf, as a percentage of the corresponding growth rate of the wild-type strain ( 0.32 h)1), was only 55%
Fig 3 Growth rates Diagram showing the growth rate of strain
GRd.tsf as a function of the growth rate of the wild-type strain UY211.
Each point represents a specific medium either based on Luria–Bertani
(LB) medium (j) or supplemented minimal medium (h).
Fig 4 Amino acid starvation and guanosine 5¢-diphosphate 3¢-diphosphate (ppGpp) and guanosine 5¢-triphosphate 3¢-diphosphate (pppGpp) [(p)ppGpp] levels (A) Growth curves of Escherichia coli UY211 (s) and GRd tsf (j) in glucose/Mops initially containing 40 l M of proline.(B) (p)ppGpp levels in cultures of UY211 (ppGpp, s; pppGpp, d) and GRd.tsf (ppGpp, h; pppGpp, j) during proline exhaustion presented in relation to the attenuance D 420 of the cultures shown in (A).The levels of (p)ppGpp were normalized to cell densities and are presented relative to the level of pppGpp in UY211 before starvation, giving this level a value of 1.
Trang 7Guanine nucleotide exchange and poly(U)-directed
poly(Phe) synthesis
The ability of the deletion mutant of EF-Ts to stimulate the
activity of EF-Tu was tested in guanine nucleotide exchange
and poly(U)-directed poly(Phe) synthesis assays (Fig.6)
The activity of the mutant EF-Ts in promoting the exchange
of EF-Tu-bound GDP with free [3H]GDP was 8.5 pmol
exchanged GDP/(min· pmol EF-Ts) and, thus, 75% of
the activity of the wild-type EF-Ts [11.1 pmol exchanged
GDP/(min· pmol EF-Ts)] (Fig.6A).When the nucleotide
exchange activity was measured using [3H]GTP instead of
[3H]GDP, the activity of the deletion mutant of EF-Ts was
65% of the activity of the wild-type EF-Ts (data not
shown).However, the deletion mutant of EF-Ts was as
active as the wild-type EF-Ts in promoting poly(U)-directed
poly(Phe) synthesis with EF-Tu under the conditions used
(Fig.6B).The activity in the translation assay was 6.2 pmol
[14C]Phe incorporated/(min· pmol EF-Ts) for both EF-Ts
species
Dissociation of the EF-Tu–EF-Ts complex by guanine nucleotides and kirromycin
The stability of the mutant EF-Tu–EF-Ts complex in the presence of guanine nucleotides and kirromycin was analysed using zone-interference gel electrophoresis (Fig.7) The principle of zone-interference gel electro-phoresis, described by Abrahams et al.[45], was used for the analysis to ensure that the concentrations of ligands were maintained at a constant level during migration of the elongation factors, thereby favouring equilibrium conditions.The concentration of guanine nucleotides required to dissociate the mutant EF-Tu–EF-Ts complex was at least two orders of magnitude lower than that for the wild-type complex.While the wild-type EF-Tu–EF-Ts complex remained stable at 1 mMGDP (Fig.7A, lane 8), the mutant complex dissociated at 10 lM GDP (Fig.7A, lane 14).When the experiment was repeated with GTP instead of GDP, the wild-type EF-Tu–EF-Ts complex remained stable at 10 mM GTP (Fig.7B, lane 7), while the mutant complex dissociated at 0.1 mMGTP (Fig.7B, lane 11).Unfortunately, concentrations of guanine nucleo-tides higher than 10 mMcaused band smears, which made
it impossible to detect dissociation of the wild-type EF-Tu–EF-Ts complex
The stability of the mutant EF-Tu–EF-Ts complex, in the presence of kirromycin, was examined at 10 lM GTP or
1 lM GDP, as these concentrations did not cause any dissociation of either the wild-type or the mutant EF-Tu– EF-Ts complex in the absence of kirromycin (Fig.7B,A, respectively).In the presence of 5 lMkirromycin, the wild-type complex dissociated (Fig.7C, lane 10), while a small, but reproducible, bandshift was observed with the mutant complex (Fig.7C, lane 15).The bandshift was thought not to
be related to dissociation, as this very dense band migrated faster than any of the individual components (compare lanes
3, 4, 6 and 15 in Fig.7C).When the experiment was repeated with 1 lMGDP (Fig.7D), instead of 10 lMGTP, the wild-type complex still dissociated at 5 lMkirromycin (Fig.7D, lane 8), while the mutant complex appeared to dissociate at
Fig 5 Growth in the presence of kirromycin The growth rate of strain
GRd.tsf is presented as a percentage of the corresponding growth rate
of the wild-type strain, UY211, in the presence of kirromycin.
Fig 6 Stimulation of the activities of elongation factor Tu (EF-Tu) by the coiled-coil deleted elongation factor Ts (EF-Ts) mutant (A) Stimulation of the exchange of EF-Tu-bound GDP with free [ 3 H]GDP by wild-type EF-Ts (s) and the coiled-coil deleted EF-Ts mutant (j).(B) Stimulation of the activity of EF-Tu in poly(U)-directed poly(Phe) synthesis by wild-type EF-Ts (s) and the coiled-coil deleted EF-Ts mutant (j).The nucleotide exchange assays were repeated at least five times and the poly(Phe) assay was repeated six times.Each point in the figure represents a slope obtained from a time curve containing six measurements.The data shown are representative of the results obtained on each occasion.
Trang 850 lMkirromycin (Fig.7D, lane 18).In this experiment, no
bandshift was observed
Discussion
Phenotype ofE coli mutant GRd.tsf
The results presented in Fig.3 show that the functional
activity of the coiled-coil motif of EF-Ts does not limit
growth at growth rates below 0.8 h)1.However, in media
supporting higher growth rates, the mutant strain appears
to be impaired in a proportional and medium-dependent
manner over all growth rates.This result is somewhat
surprising because growth conditions studied often consist
of restricted media, in which the concentration of growth
substrates is limited, rather than investigating the ability of
cells to take up and utilize an unlimited concentration of
growth substrates.The molar ratio of EF-Tu to ribosomes,
as well as to EF-Ts, is known to decrease as the growth rate
increases.In contrast, the ratio of EF-Ts to ribosomes
is maintained at 1 : 1, irrespective of the growth rate
[46–48].Therefore, it is reasonable to assume that an
increase in the growth rate and, thus, in the rate of protein
synthesis, requires a faster recycling of EF-Tu.Hence, the
concentration of EF-Tu–GDP probably increases with an
increase in growth rate, and thereby the requirement for
EF-Ts activity will also increase.Based on this assumption, the present results indicate that the maximal rate of the mutant EF-Ts in protein synthesis in vivo is 70% of that of the wild-type EF-Ts
The studies of the effect of the EF-Ts mutation under starvation conditions suggest that the growth inhibition of GRd.tsf at a low cell density is caused by the binding of a deacylated tRNA to the ribosome rather than by a change in the synthesis of (p)ppGpp.Deacylated tRNA has a high codon-specific affinity for the ribosomal A site [49].A moderate decrease in the level of active EF-Tu– GTP, owing to a less efficient EF-Ts, could cause ribosomal binding of deacylated tRNA and thereby (p)ppGpp synthesis at a lower cell density.This is in accordance with the view of Glazier et al.[50], who monitored the synthesis of (p)ppGpp in the temperature-sensitive EF-Ts mutant strain, HAK88, of E coli.There-fore, the present results indicate that the deletion of the coiled-coil motif in EF-Ts probably reduces the formation
of EF-Tu–GTP–aa-tRNA owing to a decrease in the regeneration of EF-Tu–GTP
A complete deletion of the tsf gene in bacteria has, to our knowledge, never been reported.In comparison, deletion of the single-copy gene, TEF5, in yeast, which encodes the
Fig 7 Analysis of the elongation factor Tu (EF-Tu)–elongation factor Ts (EF-Ts) complex in the presence of guanine nucleotides and kirromycin by zone-interference gel electrophoresis (A) Wild-type and mutant EF-Tu–EF-Ts complexes in the presence of different concentrations of GDP (0–1.0 m M ).(B) Wild-type and mutant EF-Tu–EF-Ts complexes in the presence of different concentrations of GTP (0–10 m M ).(C) Wild-type and mutant EF-Tu–EF-Ts complexes in the presence of 10 l M GTP and different concentrations of kirromycin (0–50 l M ).The bandshift described in the text is indicated by a star.(D) Wild-type and mutant EF-Tu–EF-Ts complexes in the presence of 1 l M GDP and different concentrations of kirromycin (0–50 l M ).Sample solutions contained 50 pmol EF-Tu–EF-Ts complex.
Trang 9guanine nucleotide-exchange factor (eEF1Ba)
correspond-ing to EF-Ts, is lethal [51].Thus, the observed growth
reduction of up to 70% for the mutant EF-Ts in this study
probably relates specifically to the deletion of the coiled-coil
motif.We predict that also deletion of the tsf gene would be
lethal
Guanine-nucleotide exchange activity
The ability of EF-Ts to promote the guanine nucleotide
exchange in EF-Tu was reduced by the deletion of the
coiled-coil motif.This result supports the view that the lower growth
rate of strain GRd.tsf, and its earlier synthesis of (p)ppGpp
during amino acid exhaustion, are caused by a reduction in
the rate of guanine nucleotide exchange
However, even though much effort was put into
optimi-zing the poly(U)-directed poly(Phe) synthesis assay to ensure
that the guanine nucleotide exchange in EF-Tu limited the
polymerization, no effect of deleting the coiled-coil motif in
EF-Ts was detected in this assay.Thus, the polymerization
assay appeared to be less sensitive than the guanine
nucleotide-exchange assay, as previously reported [52]
Stability of the EF-Tu–EF-Ts complex in the presence
of guanine nucleotides
The stability of the mutant EF-Tu–EF-Ts complex in the
presence of guanine nucleotides was examined by the use of
zone-interference gel electrophoresis (Fig.7A,B).The fact
that the concentration of guanine nucleotides required to
dissociate the mutant complex was at least two orders of
magnitude lower than for the wild-type complex shows that
the deletion of the coiled-coil motif shifts the equilibrium in
the guanine nucleotide-exchange reaction towards
dissoci-ation of the EF-Tu–EF-Ts complex.This result suggests
that the reduced activity of the mutant EF-Ts in guanine
nucleotide exchange is caused by a reduction in the ability to
compete with guanine nucleotides for the binding to EF-Tu
Previous mutational studies of E coli EF-Ts, concerning
residues directly involved in the binding of EF-Tu, have
indicated a strong correlation between the abilities of the
mutated forms of EF-Ts to compete with GDP for binding
to EF-Tu and their activities in promoting guanine
nucleo-tide exchange [53].In contrast, the present results indicate
that deletion of the coiled-coil motif in EF-Ts, which is not
in contact with EF-Tu in the EF-Tu–EF-Ts complex
(Fig.1), strongly reduces the ability of EF-Ts to compete
with GDP as well as GTP for binding to EF-Tu.Moreover,
the activity in promoting guanine nucleotide exchange is
only slightly reduced
Stability of the EF-Tu–EF-Ts complex in the presence
of kirromycin
The growth of GRd.tsf in the presence of kirromycin was
examined because the binding of kirromycin and EF-Ts to
EF-Tu has been shown to be mutually exclusive [27].In the
presence of less than 0.5 lM kirromycin in the medium,
strain GRd.tsf was unaffected, while at concentrations
higher than 0.5 lMkirromycin, the growth rate of GRd.tsf
was reduced to a greater degree than that of the wild-type
strain.This suggests that the competition between
kirro-mycin and EF-Ts for the binding of EF-Tu is altered by deletion of the coiled-coil motif
Examination of the EF-Tu–EF-Ts complex by zone-interference gel electrophoresis in the presence of kirromycin supported that the mutant EF-Ts has an altered ability to compete with kirromycin for the binding of EF-Tu.In the presence of 10 lMGTP and 5 lMkirromycin, the mutant EF-Tu–EF-Ts complex had a slightly higher mobility than the mutant EF-Ts, the mutant EF-Tu–EF-Ts complex in the absence of kirromycin, and also the EF-Tu–GTP– kirromycin complex.Therefore, the observed bandshift cannot be explained by dissociation of the mutant EF-Tu– EF-Ts complex.This could indicate that a higher-order complex containing kirromycin might have been formed The mobility shift of the mutant complex occurs at the same concentration of kirromycin (5 lM) required to dissociate the wild-type complex.It is reasonable to expect that kirromycin will bind wild-type EF-Tu at the same concen-tration in both experiments.Therefore, we suggest that the shifted complex of the mutant EF-Tu–EF-Ts is EF-Tu– GTP–kirromycin–EF-Ts.The observed moderate increase
in mobility of the complex in the presence of GTP and kirromycin is in accordance with what would be expected by the binding of a small (794 Da) and weakly acidic ligand, such as kirromycin [54,55], assuming that the binding of kirromycin to the mutant EF-Tu–EF-Ts complex does not induce large conformational changes
Stabilization of the mutant EF-Tu–EF-Ts complex by the binding of kirromycin would reduce the pool of EF-Tu and EF-Ts available for protein synthesis in the cell.This could explain the additional reduction in the growth rate of GRd.tsf compared with that of the wild-type strain in the presence of the antibiotic.The formation of a quaternary complex would probably require a conformational change
in the EF-Tu–EF-Ts complex, as the binding site of kirromycin is not present in the EF-Tu–EF-Ts structure
In addition, the structure of the wild-type EF-Tu–EF-Ts complex [9], and that of the complex between EF-Tu–GDP and aurodox (methyl kirromycin) [25], show that the binding sites of EF-Ts and kirromycin on EF-Tu are not overlapping.Thus, the suggested binding of kirromycin to the mutant EF-Tu–EF-Ts complex either indicates that the conformation of EF-Tu in the mutant complex is different from that of the wild-type complex, or that the mutant complex is more flexible than the wild-type complex, which might facilitate the binding of the antibiotic
Comparison of mutant EF-Ts and EF-Ts from bovine mitochondria
Elongation factors equivalent to the prokaryotic EF-Tu and EF-Ts are active during protein synthesis in mitochondria and chloroplasts.The coiled-coil motif is preserved in prokaryotes and chloroplasts, but absent in mammalian mitochondria.Therefore, it was of interest to compare the activities of mutant EF-Ts and EF-Tsmt.However, such a comparison should be undertaken bearing in mind the many differences between the two EF-Ts species.EF-Tsmtis only 29% identical to E coli EF-Ts.Furthermore, EF-Tsmt has an 11-residue C-terminal extension, 21 residues inserted between helices 5 and 6, and 12 residues inserted between b-strands 4 and 5, compared with E coli EF-Ts
Trang 10Unlike the EF-Tu–EF-Ts complex from E coli, the
corresponding mitochondrial complex is not easily
disso-ciated by nucleotides [56].When EF-Tsmt is expressed in
E coli, it forms a heterologous complex with the
endo-genous EF-Tu, which is 100-fold stronger than the
homologous E coli complex.The heterologous complex
has been found to be active in poly(U)-directed protein
synthesis [57,58].It is mainly the region in EF-Tsmt,
corresponding to subdomain N in the core domain of
E coli EF-Ts, that appears to be responsible for the
strong complex formation with E coli EF-Tu [58,59].Our
studies have shown that the coiled-coil motif also plays a
central role in controlling the stability of the
EF-Tu–EF-Ts complex.In contrast to the enhancing effect of the
mitochondrial subdomain N on the strength of a
hetero-logous EF-Tu–EF-Ts complex, the effect of making the
E coli EF-Ts more mitochondrial-like by deleting the
coiled-coil region is a dramatic weakening of the complex
Therefore, one would expect that an EF-Ts chimera
between the N-terminal half of EF-Tsmt and the
C-terminal half of E coli EF-Tu should form an even
stronger complex with E coli EF-Tu.This turns out to be
the case, as shown previously by Zhang et al.[58]
Previous studies have shown that mutations in EF-Tsmt,
which cause a weakening in the interaction with E coli
EF-Tu, correlate with an increased ability to stimulate GDP
exchange as well as an increased activity in polymerization
[53,58].The authors suggest that the strong interaction
between EF-Tsmtand E coli EF-Tu makes it difficult for
guanine nucleotides to compete for interaction with EF-Tu,
thereby reducing the stimulatory effect of EF-Ts.Therefore,
the effect observed was probably caused by a shift in the
equilibrium governing the exchange reaction.We did not
observe a similar correlation in our studies, as both the
strength of the EF-Tu–EF-Ts complex, as well as the
stimulatory effect on guanine nucleotide exchange, was
decreased by deletion of the coiled-coil motif.This might
indicate that the coiled-coil motif is directly involved in
guanine nucleotide exchange
The role of the coiled-coil motif
The results of the present study demonstrate that the
coiled-coil motif in E coli EF-Ts is not crucial for the
stimulatory effect of EF-Ts on the guanine
nucleo-tide exchange in EF-Tu, even though the stability of the
EF-Tu–EF-Ts complex in the presence of guanine
nucleotide is dramatically reduced.Although the
coiled-coil motif is not strictly required for survival, it confers a
strong selective advantage to a cell; this explains its
preservation during evolution.In support of this, the
coiled-coil motif has apparently been lost from the
mitochondria after its origin as an aerobic bacterium that
established residency within the cytoplasm of a primitive
eukaryote (according to the endosymbiotic theory) [60].In
this manner, the eukaryotic partner was supplied with
energy in exchange for a stable, protected environment,
and a readily available supply of nutrients.In a milieu
such as this, the ingested bacterium would have no
advantage for preserving the coiled-coil motif
Previous studies of E coli EF-Ts derivatives [52,53], in
combination with the present results, suggest that the most
essential regions for the activity of EF-Ts are located in the N-terminal domain and the core domain.These observa-tions are compatible with the absence of both the coiled-coil motif and the C-terminal module in mammalian EF-Tsmt, which can bind E coli EF-Tu and stimulate the activity of this elongation factor [31,57]
The present results support the view that the E coli EF-Tu–EF-Ts complex exists as a heterodimer, rather than as a heterotetramer in translation, and therefore indicate that the crystallographic dimerization of E coli EF-Ts is only caused by the packing of the EF-Tu–EF-Ts complex in the crystals.In contrast to E coli EF-Ts, the protruding antiparallel coiled-coil motif (helices 6 and 7)
of Thermus thermophilus EF-Ts is not involved in any dimerization in the crystal structure of the EF-Tu–EF-Ts complex [28].However, the dimerization of the coiled-coil motifs in the E coli EF-Tu–EF-Ts crystals suggests an affinity of the coiled-coil motif for an unidentified helical structure.In this regard, the present results could indicate that such a helical structure interacts with the coiled-coil motif during a conformational change in one step of the nucleotide exchange reaction and somehow stabilizes the EF-Tu–EF-Ts complex in the presence of guanine nucleotides
Pressure relaxation studies of the nucleotide exchange reaction have indicated that the ternary complex, EF-Tu– thioGDP–EF-Ts, undergoes an isomerization step [7] Likewise, Gromadski et al.[1] have proposed that the binary complex, EF-Tu–EF-Ts, and the ternary complexes containing EF-Tu, EF-Ts and GDP/GTP, are structurally different.Inspection of the structure of E coli EF-Tu– EF-Ts suggests that the N-terminal domain of EF-Ts, which consists of helical structures, might be a good candidate as an interaction partner for the coiled-coil motif The N-terminal domain appears to be highly mobile and disordered in free EF-Ts [28], and is separated from the core domain by a highly accessible region [38].Based on the present results, we propose that the preserved antiparallel coiled-coil motif in bacterial EF-Ts is involved in a protein– protein interaction within the EF-Ts molecule during an isomerization step of the nucleotide-bound EF-Tu–EF-Ts complex.Two-stranded antiparallel coiled-coil motifs are known to be involved in either protein–protein interactions [61,62] or protein–RNA interactions [63].The proposed intramolecular interaction may be an integrated step in the guanine nucleotide-exchange mechanism
Acknowledgements
We would like to thank Karen Margrethe Nielsen and Lene Kristensen for technical help and Drs Gregers Rom Andersen and Jens Nyborg for valuable discussions and ideas.Financial support from the Program for Biotechnological Research of the Danish Natural Science Research Council is gratefully acknowledged.
References
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2.Krab, I M.& Parmeggiani, A.(1998) EF-Tu, a GTPase odyssey Biochim Biophys Acta 1443, 1–22.