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Bioscience Reports 4, 917-923 (1984)
Printed in Great Britain
Nucleotide
917
sequence of the t h i o r e d o x i n
gene
from Escherlchia coli
o~
,,
3an-Olov HOOG 1, Hedvig VON BAHR-LINDSTROM 1,
Staffan 3OSEPHSONI~ Betty 3. WALLACE~, Sidney R. KUSHNER 3,
Hans 3ORNVALLI~ and Arne HOLMGREN 1
iDepartment of Chemistry I, Karolinska Institutet,
S-I04 01 Stockholm; 2Department of Chemistry, KabiGen AB,
S-I12 87 Stockholm, Sweden; and 3Department of
Molecular and Population Genetics, University of Georgia,
Athens, GA 30602, U.S.A.
(Received 27 September 1984)
The nucleotide sequence of the thioredoxin gene from
c o l i was determined.
The structural gene
was i d e n t i f i e d on a cloned 3-kb PvuII Iragment by
hybridization with a synthetic oligodeoxyribonucleotide
corresponding to a part of the amino acid sequence of
thioredoxin.
Restriction-enzyme fragments were used
as templates in the dideoxy sequence method, directly
and after subcloning into M13mpS. A segment of 450
nucleotides was determined using both strands7 alternatively~ w i t h o u t e x t e n s i v e o v e r l a p s .
The sequence
c o n t a i n s the t h i o r e d o x i n coding region~ a potential
ribosome-binding site~ and a putative promotor region.
The p r e d i c t e d a m i n o acid sequence differs by two
i n v e r s i o n s from the p r e v i o u s l y given t h i o r e d o x i n
sequence.
The revised sequence is presented and the
results further show that thioredoxins from E. c o l i B
and K12 are identical.
Escherichia
Thioredoxin from Escherichia coli has suggested functions as a
general protein disulphide reductase (1) and as a hydrogen donor for
ribonucleotide reductase (1) and is also an essential subunit of phage
T7 DNA polymerase (2).
T h i o r e d o x i n from E. c o l i consists of 108 amino acid% and a
primary structure for this protein has been deduced (3). The t e r t i a r y
structure has also been determined (4) as well as the relationship to
g l u t a r e d o x i n (5-7) and i n t r a c e l l u l a r localizations (8).
Mapping
experiments have located the gene for thioredoxin~ t r x A , at 84 min on
the E. c o l i chromosome (9).
Recently~ a segment of the E. c o l i
chromosome containing the thioredoxin gene has been ]igated into the
ptasmid pBR325 (10).
The resulting plasmid pBHK8 transformed into
E.
coli
K12 ( s t r a i n : 5K3981) o v e r p r o d u c e s thioredoxin 150- to
200-fold ( I 0 ) .
01984
The Biochemical Society
918
HDOG ET AL.
Knowledge of the nucleotide sequence of the thioredoxin gene will
e n a b l e s t u d i e s using site-directed mutagenesis to define structurefunction relationships of thioredoxin.
In this study9 we report determination of the nucleotide sequence
of the thioredoxin gene from E. c o l i 9 independent of similar studies
elsewhere (11) 9 we analyze the protein~ and compare the amino acid
prediction from DNA with the primary structure of thioredoxin.
i'
M a t e r i a l s and Methods
Preparation of DNA
The plasmid pBHK8 was isolated from E. coli SK3981 with the
alkaline lysis method (10912). Digestions of plasmid DNA by restriction endonucleases were performed using the conditions suggested by
the manufacturer (Boehringer).
Restriction endonuclease fragments
w e r e s e p a r a t e d by a g a r o s e - g e l e l e c t r o p h o r e s i s and i s o l a t e d by
electro-elution in dialysis bags (12).
Synthesis of oligodeoxyribonucleotides
O l i g o d e o x y r i b o n u c i e o t i d e s w e r e s y n t h e s i z e d by the solid-phase
phosphoamidite method using an automatic synthesizer (13914).
The
concentrations
of t h e f o u r 5 ' - O - d i m e t h o x y t r i t y l - 2 ' - d e o x y nucleoside-3'-O-methyl phosphodimethylamidite reagents were measured
(1#) prior to making the mixtures so as to get equal ratios between
reagents.
Initially 9 a mixed l#-mer was synthesized corresponding to
the known amino acid sequence (3). When part of the DNA structure
was known 9 a 15-mer was synthesized corresponding to the nucleotide
sequence.
Hybridization with synthetic oligodeoxyribonucleotides
Oligodeoxyribonucleotides (0.01 pg/pl) were labelled at the 5' end
by transfer from [y-32P]ATP (Amersham; 3000 Ci/mmol; 1:2 molar
ratio [y-g2P]ATP:oligodeoxyribonucleotides) by using T# polynucleotide
kinase (New England Biolabs; 2 U/pl) in 50-ram Tris/HCl pH 7.59
10-ram MgCI29 10-ram dithiothreitol for #5 rain at 37~
Excess
reagents were removed by separation on Sephadex G-50 (0.25 x 15
cm) in 10-ram Tris/HCl, l-raM EDTA9 pH 7.5. Radioactively labelled
oligodeoxyribonucieotides (3 x 108 c . p . m . / p g ) w e r e used as probes for
hybridization analysis by the Southern blot (15916) and dot-blot (17)
techniques.
Nucleotide sequence and protein analysis
S a u 3 A fragments were cloned into the BamHI site of Ml3mpg (18).
Transfection of E. c o l i 3M 103 was performed as described (19).
Sequence analysis by the dideoxy method (20921) utilizing singlestranded M13-templates (22) and restriction-enzyme fragments after
heat-induced strand separation (23) was carried out with either an
Mi3-specific universal primer (Amersham) or with the thioredoxinspecific oligodeoxyribonucleotides as primers.
The labelled nucleotide
was [~-aSS]dATP (Amersham~ 600 Ci/mmol) and the reaction mixture
was s e p a r a t e d by electrophoresis in a 0.2-ram-thick 6% urea-polyacrylamide gel.
NUCLEOTIDE SEQUENCE OF E. COLI THIOREDOXIN GENE
A
919
B
3 kb
Fig. I.
Identification of the fragment from pBHK8
containing the thioredoxin gene. Lane A: Ethidium
bromide stain after agarose-gel electrophoresis of a
PvuII digest of pBHK8, showing the 3-kb and 2.6-kb
fragments (I0).
Lane B: Autoradiograph of lane A
after transfer to nitrocellulose by the method of
Southern (15) and hybridization with the 32p_
labelled mixed 14-mer oligodeoxyribonucleotide
(Table i).
SK3981 was prepared as described (10)
a n a l y s i s was carried out with the DABITC
Thioredoxin from E. coli
and manual s e q u e n c e
method (24925).
Results
The plasmid pBHK8 was cleaved with z~zuII and the digest was
separated by agarose-gel electrophoresis (Fig. 1).
A 3-kb fragment
containing the thioredoxin gene was identified (Fig. 1) after transfer
to nitrocellulose by the method of Southern and hybridization with the
mixed 14-mer synthetic probe (Table 1).
The 3-kb fragment was
isolated from agarose gel and used for sequence analysis directly and
after subcloning into Ml3mp8.
Table I. Synthetic oligodeoxyribonucleotides used as
hybridization probes and primers in dldeoxy sequence analysis
28
Amino acid
sequence
-
Trp
32
-
Ala
-
Glu
-
Trp
-
Cys
-
C
14-mer
3"
A C C C G T C T ~ A C C A C
5"
A
52
57
- Lys - Leu - T h r - Va] - A l a - Lys
Amino a c i d
sequence
15-mer
5"
ACTGAC
CGTTGCAAA
3"
920
Hb6G
50
I
A
I
i
100
100
I
i
200
ET AL.
l
300
-I
400
C3
ED
Fig. 2. Summary of nucleotide sequence data.
Line
A represents
the complete structure determined,
with amino acid sequence numbering above the line
and nucleotide sequence numbering below.
Line B
represents the two Sau3A fragments sequenced in the
MI3 system.
L i n e s C and D show the regions
sequenced directly from the 3-kb fragment using as
primer, respectively,
the mixed 14-met and the
15-mer oligodeoxyribonucleotides (Table I).
The 3-kb f r a g m e n t was digested with Sau3A, and the mixture
o b t a i n e d was cloned i n t o M l 3 m p g .
A # g - n u c l e o t i d e fragment9
i d e n t i f i e d as c o m p l e m e n t a r y to the mixed l#-mer probe by the
dot-blot hybridization technique, was sequenced (Fig. 2).
Based on
the sequence of another Sau3A fragment (Fig. 2) from the thioredoxin
gene~ a 15-mer oligodeoxyribonucleotide was synthesized (Table 1) for
use as a primer in the sequence analysis.
The isolated 3-kb fragment was used as template in direct dideoxy
sequence analysis after heat-induced strand separation.
The mixed
l#-mer oligodeoxyribonucleotide was utilized as primer to determine
the nucleotide sequence of the coding strand on the 5' side of the
r e g i o n c o m p l e m e n t a r y to the primer (Fig. 2) (26).
The 15-mer
oligodeoxyribonucleotide was used in the same way to determine the
sequence of the non-coding strand on the 3' side of the oligodeoxyr i b o n u c l e o t i d e (Fig. 2).
The resulting nucleotide sequence of the
thioredoxin gene is shown in Fig. 3.
The N-terminal sequence of thioredoxin from E. eo2i strain SK39gl
was d e t e r m i n e d
w i t h t h e D A B I T C method and found to be
Ser-Asp-Lys-lle-~ which is identical to that of E. co2i strain B (3).
Discussion
The n u c l e o t i d e sequence of the thioredoxin gene from E. coli K12
is given in Fig. 3 t o g e t h e r with the p r e d i c t e d amino acid sequence.
The predicted
protein structure
was f o u n d to d i f f e r from the
p r e v i o u s l y r e p o r t e d amino a c i d sequence (3) at two places.
The
d i f f e r e n c e s c o n c e r n positions t6-17 and 71-72, which have been quoted
as Leu-Val and Ile-Gly~ r e s p e c t i v e l y (Fig. 5 in ref. 3).
However,
t h e s e s t r u c t u r e s were mis-quotations of original protein data~ giving
Val-Leu (27) and Gly-Ile (Fig. # in ref. 3)~ and the orders are now
v e r i f i e d and established by the present n u c l e o t i d e sequence.
The
e n t i r e gene was not analyzed on both strands.
This was considered
u n n e c e s s a r y since the n u c l e o t i d e sequence a g r e e s with the amino acid
sequence.
NUCLEOTIDE SEQUENCE OF E. COLI THIOREDOXIN GENE
TCA GCT TAA CTA TTG CTT TAC GAA AGC GTA
TTT AGT
TGG TTA ATG
CCT G T ~ G A G T T ~
TCC GGT GAA ATA AAG TCA ACC
TTA CAC CAA CAA CGA AAC CAA CAC GCC AGG
TAT ATG AGC
GAT AAA ATT ATT
Ser Asp Lys
Ile
Ile
Leu Thr Asp Asp Ser
10
TTT GAC ACG GAT GTA CTC AAA GCG
Val Leu Lys
CTT ATT
CAC CTG ACT GAC GAC AGT
His
1
Phe Asp Thr Asp
921
GAC GGG GCG ATC CTC GTC GAT
Ala Asp Gly Ala
TTC TGG
Ile Leu Val Asp P h e Trp
20
GCA GAG TGG TGC GGT CCG TGC AAA ATG ATC GCC CCG ATT CTG GAT GAA ATC
AIa GIu Trp s
Giy Pro Cys Lys Met Iie A1a Pro lie Leu Asp Giu Ile
30
40
GCT GAC GAA TAT CAG GGC AAA CTG ACC GTT
A1a
GCA AAA CTG AAC ATC GAT CAA
Asp G1u Tyr Gln G1y Lys Leu Thr Val Ala
Lys Leu Ash
50
GO
AAC CCT GGC ACT GCG CCG AAA TAT GGC ATC
Asn
Pro Gly
Thr Ala
lie Asp Gin
Pro tys
Tyr Gly
CG~ GGT ATC CCG ACT CTG CTG
lie Arg Gly
lie Pro Thr Leu Leu
70
CTG
TTC AAA AAC GGT GAA GTG GCG
GCA ACC AAA GTG GGT GCA CTG TCT AAA
Leu
Phe Lys Ash Gly Glu Val Ala
Ala Thr Lys Val Gly Ala Leu
80
Ser Lys
9O
GGT CAG TTG AAA GAG TTC CTC GAC GCT AAC
CTG GCG
Gly
Leu Ala
Gin Leu
Lys Glu
100
Phe Leu Asp
Ala Ash
TAA GGG AAT
108
Fig. 3. Nucleotide sequence of the thioredoxin gene
and its corresponding amino acid sequence.
The
nucleotide sequence of 450 nucleotides comprising
the non-coding strand of the thioredoxin gene is
s h o w n in the 5'§
direction.
The proposed
ribosome-binding site is boxed and the -35 and -I0
sites of the proposed promotor are indicated by
lines below the nucleotide sequence.
The E . c o l i
KI2 strain SK3981 containing the plasmid pBHK8
produces thioredoxin in amounts great er than 300 mg per litre of
culture medium (10), which is 150-200 times more than wild-type E.
coli
B.
A normal processing of the thioredoxin N-terminus by the
o v e r p r o d u c i n g strain is indicated by the N-terminal serine residue,
which is identical to that of the wild-type protein.
This contrasts
with N - t e r m i n i d e t e r m i n e d for glutaredoxin, a small redox-protein
belonging to the same superfamily as thioredoxin (5). The glutaredoxin
from the overproducing E . c o l i K12 strain C[0-17 has an N-terminal
methionine residue, followed by a glutamine residue (5), compared to
the protein from E. coli B which starts with glutamic acid/glutamine
(28).
Altogether, the nucleotide sequence determination and the
presently determined N-terminal amino acid sequence compared with
the previously known amino acid sequence shows that there is no
strain variation for thioredoxin from E. coli B and KI2 strain SK3981.
922
HOC)G ET AL.
The thioredoxin coding region is initiated by an AUG triplet. It is
preceded by a potential ribosome-binding site (G-G-A-G-U-U) showing
f i v e out of six residues identical to the Shine-Dalgarno sequence
(G-G-A-G-G-U) (29).
The coding region is terminated by an ochre
(U-A-A) codon.
The best putative promotor region is 70 base pairs
upstream of the translation-initiation codon.
It comprises a Pribnow
box and a -35 region (Fig. 3); both showing four out of six residues
i d e n t i c a l to the respective consensus sequences (30) in E. c o l t .
Therefore, it seems as if the transcription of E. c o l t thioredoxin is
governed by its own promotor region, which is consistent with the fact
that thioredoxin is an abundant protein (104 copies/cell (31)).
The
results also show that thioredoxin is not synthesized with a signal
sequence, although it is localized in the periphery of E. co.Zi cells
(~).
During the identification and nucleotide sequence determination of
a plasmid containing the rho gene from E. colt K12, the presence of
an unidentified, expressed polypeptide of 12 000-14 000 Da was
observed (32). The sequence of the first 60 nucleotides of the 390
preceding the coding part of the rho gene (32) is identical to the last
residues of the coding part of the thioredoxin gene (Fig. 3). This
identifies the unknown polypeptide as thioredoxin and furthermore
locates the gene for thioredoxin upstream of the rho gene in E. eoli
K12.
Acknowledgements
We thank Drs. Lars Hellman~ Erik Holmgren, and Lars Wieslander
for v a l u a b l e discussions and Eva Lagerholm for synthesis of the
oligodeoxyribonucleotides.
This work was s u p p o r t e d by grants from the Swedish Cancer
S o c i e t y , the Swedish Medical Research Council, Magn. Bergvall's
Foundation, and the National Institute of Health (6m27997).
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NUCLEOTIDE SEQUENCE OF E. COLI THIOREDOXIN GENE
923
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