Download Nucleotide sequence of the Tn10 encoded tetracycline resistance

volume 11 Number 2 1983
Nucleic Acids Research
NndeotMe sequence of tbe TnlO encoded tetracydine resistance gene
Wolfgang Hillen and Klaus Schollmeier
Institut far Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22,
D-6100 Darmstadt, FRG
Received 29 September 1982; Revised 26 November 1982; Accepted 2 December 1982
ABSTRACT
The nucleotide sequence of 1530 base pairs of TnlO DNA
coding for tetracycline resistance has been determined. The
gene start consists of overlapping bidirectional promotors and
two operator sequences. One terminator of transcription as defined by the typical terminator sequence is about 1300 base
pairs downstream from the promotor. It is preceeded by translation termination codons in all three possible reading frames.
The transcript contains an open reading frame coding for a 4 3.3
kDa protein. Two other possible reading frames are discussed.
The amino acid sequence of the Tn10 encoded tetracycline resistance gene shows good homology with two proteins encoded in the
tetracycline resistance part of the plasmid pBR322. The hydrophobic nature of the 43.3 kDa protein is discussed with regard
to it's proposed function.
INTRODUCTION
The tetracycline resistance determinant of the transposon
Tn10 is located on a 2700 bp Hpa I fragment [1]. A reoressor
protein controling expression of the resistance gene is encoded
on the leftward 700 bp [2]. The promotor and operator reaion
for the repressor and resistance proteins consists of two bidirectional overlapping promotors and two operators [2,3]. The
TET repressor has been isolated and the mechanism of regulation
of the tetracycline resistance evaluated at a molecular level
[4]. In addition to the 25 kDa TET repressor, a tetracyclineinducible 36 kDa resistance protein was detected in minicell
assays, encoded by a gene which runs in the opposite direction
to that encoding the repressor [2]. In addition, a tetracyclineinducible, low molecular weight protein has been detected on
R222, a resistance factor bearing Tn10 [5]. Recently, genetic
evidence was provided for the existence of two gene loci in the
tet resistance part of the tet gene which were called tet A and
© IRL Press Limited, Oxford, England.
03O5-1O48/83/1102-O626»2.OO/0
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Nucleic Acids Research
tet B [6). It was assumed that tet B is the 36 kDa protein because it's carboxyterminal coding region extends further than
the molecular weight would imply [1,6].
The tetracycline resistance determinants from different R
factors can be subdivided into at least four genetically different classes [7]. The tetracycline resistance gene on pBR322,
the nucleotide sequence of which is known [0], belongs to class
C whereas Tn10 is from class B [7]. It is of interest to compare
the nucleotide sequences and the amino acid sequences of these
tetracycline resistance proteins.
In this article we report the nucleotide sequence of the
Tn10 encoded tetracvcline resistance gene. Possible tetracycline
resistance proteins are discussed and compared to the tetracycline resistance of pBR322.
MATERIALS AND METHODS
Materials
Restriction endonucleases and T4 DNA ligase were purchased
from 3RL, Bethesda, Md. or isolated by a published procedure
[9]. Restriction digests were done as described [9]. | P|phosDhate was obtained from MEN chemicals, Dreieich. | P|ATP was
synthesized from ADP and | P| by a published procedure [10].
T4 polynucleotide kinase was a gift of V. Eckert and R. Heinzel.
Preparation of the DNA
Tn10 DNA was prepared from the plasraids pRT61 and pRT29
[1,2]. The 675 bp Hpa I fragment from pRT61 was subcloned into
pUR222 [11] to facilitate sequencing. PreDaration of plasmids
was as described previously [12]. DNA fragments were either
eluted from polyacrylamide gels as described [13] or purified
by RPC-5 chromatography [14].
Sequencing
The nucleotide sequence of DNA fragments was determined by
the procedure of Maxam and Gilbert [13]. Reagents were obtained
from NEN chemicals, Dreieich. The products of the strand cleaving reactions were separated on denaturing polyacrylamide gels
[13] which were 0.5 mm thick and 80 cm long. The qels were exDosed to Cronex low dose plus film from DuPont. The combination
of 12.5%, 8%, and 6% polyacrylamide gels allowed us to read ap-
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Nucleic Acids Research
proximately 400 nucleotides from one reaction in the best cases.
Generally about 300 nucleotides were readable [15]. Restriction
sites were found in the sequence by screening it using a computer program provided by L. Altschmied.
RESULTS
Sequencing strategy. Figure 1 shows the sequencing strategy used
to determine the nucleotide sequence from the Xba I site to the
Hind III site [1]. Except for a small portion around the Eco RI
site the sequence of both the plus and minus strands was determined to reduce reading errors. For the same reason each part of
the 1560 nucleotides was analyzed in at least three independent
experiments. The leftmost Hinc II site in figure 1 characterizes
the promotor-operator region for tetracycline resistance as it
is protected against cleavage both by RNA polymerase [1] and
TET repressor [4]. The numbers on the top panel of figure 1 refer to the published restriction map of Tn10 [1].
Figure 2 displays the complete nucleotide sequence from the
Hinc II to the Hind III site. The leftmost 100 bp of the sequence including the overlapping promotor-operator region were
communicated to us by K.P. Bertrand and W.S. Reznikoff [3] and
also determined during the course of this study. The sequence
was analyzed to yield the positions of cleavage sites from restriction endonucleases currently commercially available. The
position of the cleavage sites are listed in table I.
Transcriptional control signals. Figure 3 shows the promotoroperator region of the tetracycline resistance gene. It had been
12 75
675
Xbol
Figure 1 Sequencing strategy for the Tn10 encoded tetracycline
resistance gene. The lengths of the DNA on the top panel is taken from the Tn10 restriction map [1].
527
00
u,
C
T
T
T
A
C
G
T
T
T
G
C
T
T
'
T C A T T G C C G A '
T T A G G G G C A A
T T T T G C A G G A
T A A A T A T T G T
C
G
G
G
C
G
G
G
G
G
G
'
C
A
G
G
G
A
T
C
A
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'
A
G
G
A
G
T
T
T
T
T
G
C
C
C
C
T
T
A
T
T
T
G
C
C
G
T
G
T
G
T
G
G
G
G
C
T
G
T
C
J
T
G
f
G
c i A A C G T T A T T ^
G G C G T A T T G C T
G C T T G G A A A A A
i A T T A A T A G G C 1
C T T T G G A T G C ' i
I ' A C C A C C T C A G C T T C T C A A C G C G T G A A G T G L
G T T T T G G G C T ' F G G T T T A A T A G C G G G G C C T A
G A G A T T T C A C C G C A T A G T C C 6 T T T T T T A T C
^ A C T T T C C T T G T G G T T A T G T T T T G G T T C C G '
I
T
A
C
G
G
Figure 2
400
650
TOO
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1530
450
500
550
600
DNA sequence from the Hinc II site to the Hind III site as shown in figure 1 [1]
A C C A A A A A T A C A C G T G A T A A T A C A G A T A C C G A A G T A G G G G T T G A G A C
A T C G A A T T C G G T A T A C A T C A C T T T A T T T A A A A C G A T G C C C A T T T T G T
T T A T T T A T T T T T C A G C G C A A T T G A T A G G C C A A A T T C ( J : C G C A A C G G T ( J
G T G C T A T T T A C C G A A A A T C G T T T T G G A T G G A A T A G C A T G A T G G T T G G
T T C A T T A G C G G G T C T T G G T C T T T T A C A C T C A G T A T T C C A A G C C T T T ( J
C A G G A A G A A T A G C C A C T A A A T G G G G C G A A A A A A C G G C J A G T A C T G C T C
T T T A T T D C A G A T A G T A G T G C A T T T G C ^ T T T T T A G C G T T T A T A T C T G A
T T G G T T A G A T T T C C C T F F T T T T A A T T T T A T T G G C T G G T G G T G G G A T C G
T A C C T G C A T T A C A G G G A G T G A T G T C T A T C C A A A C A A A G A G T C A T G A G
G G T G C T T T A C A G G G A T T A T T G G T G A G ( L : C T T A C C A A T ( F C A A C C G G T G T
T G G C C C A T T A C T G T T T A C T G T T A T T T A T A A T C A T T C A C T A C C A A T T T
A T G G C T G G A T T T G G A T F A T T G G T T T A G C G T T T T A C T G T A T T A T T A T C
C T A T C G A T G A C C T T C A T G T T A A C C C C T C A A G C T C A G G ' G G A G T A A A C A
G A C A A G F G C T T A G T T A T T T C G T C A C C A A A T G A T G T T A T T C C G C G A A I
A A T G A C C C T C T T G A T A A C C C A A G A G G ^ C A T T T T T T A C G A T A A A G A A I J
T A G C T T C A A A T A A A A C C T A T C T A T T T T A T T T A T C T T T C A A G C T C A A T
A A G C C G C G G T A A A T A G C A A T A A A T T G G C C T T T T T T A T C G G C A A G C T C
T A G G T T T T T C G C A T G T A T T G C G A T A T G C A T A A A C C A G C C A T T G A G T A
T T T T A A G C A C A T C A T C A T C A T A A G C T T
T
T
G
T
T
G
C
T
T
A
A
A
A
G
T
G
A
G
T
A
T
G
G
A
T
T
A
T
T
A
G
G
G
T
A
C
C
T
A
G A
G C
T G
T G
C T
T G
G A
A G
C T
C A
T A
G G
C'T
G G
T A
A T
A A
T T
A G
C
G
T
C
T
C C T T A T C A T G c i c
A G A T A T C G C T A A
A G G T T A T C T T T G
C G C C C A G T G C T G
G C T G G C T T T T T C
G
C
T
G
A
' j ' G G G G A T T G G
A T T G C T T C G G A
i - G C G T T A A T G d
G A T T T G G T C G G
G A T T A C T T A T +
T
A
T
A
C
G
G
T
A
T
C
T
T
C
T
C
T
T
C
C
T
T
A
C
G
G
A
T
G
G
1 5
°
200
250
300
350
T
A
C
T
C
C
G
G
G
C
T
C
T
T
G
A
G
A
C
T
G A A A A G T G A A A T G A A T A G T T C G A C A A A G A T C G C A T T G G T A A T T A C G T T A C
G
T
C
T
A
50
100
G A C A C T C T A i C A T T G A T A G A G T T A T T T T A i c A C T C C C T A T C A G T G A T A G A
>
W
3)
m
-
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Nucleic Acids Research
Table I
Position of restriction endonuclease cleavage
sites
Restriction
endonuclease
Position
Alu I
Cla I
Cfo I
Dde I
Eco RX
Eco RI
Eco RV
Fok I
Hae II
Hae III
Hga I
Hha I
Hind III
Hinf I
Hpa I
Hpa II
Hph I
Mbo I
Mbo II
Mlu I
Msp I
Pal I
Sau 3a
Sau 96 I
Sst II
Taq I
Tha I
1357, 1395, 1448, 1528
384, 429, 1233,
1206
270, 299, 337, 718
1262
426, 831 , 1235,
90, 155, 658, 7 2 2 , 735, 902, 975, 1146, 1427
657
172
344,
778,
1152
269
1 18, 358,
494,
7 3 0 , 1105, 1431
648
270, 299, 337, 718
1527
1041
1221
1094
1074 , 1275
526,
78, 375, 996
169, 857
438
1094
118, 358, 494, 730, 1105, 1431
78, 375, 996
1105
493,
1409
70, 101 , 655, 899, 1207
1294,
1410
399, 439,
demonstrated previously that transcription starts in both d i rections from a common starting area
[2-4]. The functions of
this control element are indicated in figure 3. It consists of
three promotors, one is directed towards the tetracycline r e sistance gene and two are directed towards the
tetracycline
repressor gene. The -10 homologies of two promotors overlap [16].
The third promotor is directed towards the tetracycline
repressor
gene and is 20 bp to the left in figure 3 of the overlapping
promotors. This arrangement of transcriptional start signals is
supported by in vitro transcription studies. The -10 region of
the two overlapping promotors is flanked by two palindromic sequences which serve as binding sites for the TET repressor. This
interpretation is concluded from protection and DNAse I footprint experiments
(W. Hillen, C. Gatz, and K. Schollmeier,
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Nucleic Acids Research
-35
region
Hind II
-10
region
raRNA
ATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAA
TAAAAACAACTGTGAGATAGTAACTATCTCAATAAAATGGTGAGGGATAGTCACTATCTCTTTT
raRNA
-10
""mRNA
-10,-35
-35
region
region
region
Figure 3 The tet gene regulatory region as defined by in vitro
transcription and protection experiments as well as DNAse I footprinting with TET repressor (W. Hillen, C. Gatz, and K. Schollmeier, manuscript in preparation). The -10 and -35 regions of
three promotors are indicated. The bars indicate the TET repressor binding sequences. Details will be discussed elsewhere.
manuscript in preparation) .
A typical transcription termination sequence [16] is found
around position 1320 of the sequence displayed in figure 2. The
possible secondary structure of the terminator region is displayed in figure 4. This signal is preceeded by termination
codons in all three possible reading frames within the last 80
nucleotides. Inspection of the sequence reveals the possibility
T
AT
A
A
C
C
A
C
GA T r
G
T
T
A
T
T
C
GC
G-C
C-G
T-A
T-A
T -A
A-T
T-A
5'TAGT
TAATGA
T
CATTTTTTA 3'
C-G
C-G
C-G
T-A
C-G
T-A
T-A
G-C
A
T
C
C
A A
Figure 4 Possible secondary structure of the transcription
terminator. Details are discussed in the text.
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Reading
frame
A
B
C
Sequence
Start
Stop
codon
codon
T G A T A GAG A A A A G T G A A A T G . . . TAG
T T G G A T G G A A T A G C A T G A T G , . .
TAG
G C T T C T C A A C G C G T G A A G T G . „ . TAG
Figure 5 Comparison of the Shine-Dalgarno sequences preceeding
three possible reading frames. Homologies of three or more nucleotides are underlined [18]. In addition, the start and stop
codons are shown,
of a second hairpin structure in the transcript ahead of the terminator. The stem and loop sizes are quite similar to those found
in attenuators [17], however, the possibility to form an alternative structure necessary for attenuation is not found [17]. It
is not clear whether this feature of the sequence, which is also
shown in figure 4, is of biological importance.
Possible reading frames for tetracycline resistance proteins. A
single open reading frame extends over the entire sequence and
codes for a 43.3 kDa protein. It starts 45 bases downstream from
the overlapping promotors i.e. at position 60 of the DNA sequence. A second translation start may occur at position 793 in
the same reading frame giving rise to a 17.6 kDa protein containing the 157 carboxyterminal amino acids of the 4 3.3 kDa protein.
Both of these translation starts exhibit a Shine-Dalgarno sequence preceeding the initiation codon (Fig. 5) [18]. A third
possible reading frame on the transcript starts at position 446
and terminates at 533. It lacks good Shine-Dalgarno homology and
has, thus, only a low probability to be expressed. This latter
protein would consist of 29 amino acids and have a molecular
c
|
A_
P.O
i i
HincO
ECBRI
Figure 6 Location of three possible reading frames in the tetracycline resistance part of Tn10. P,0 denotes the regulatory sequence and T shows the transcription termination signal.
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weight of 3.7 kDa. It would, however, be in a different reading
frame from the previously discussed proteins. Figure 5 compares
the DNA sequences at these possible gene starts and figure 6
gives the location of the three reading frames on the Tn10 DNA.
In the analysis to follow only the 43.3 kDa protein is considered because it completely embodies the 17.6 kDa protein, while
expression of the 3.7 kDa product is considered unlikely anyway.
Codon usage and amino acid composition„ Table II shows the result
of a codon usage analysis of the tetracycline resistance protein
Table II
Codon usage and amino acid composition of the 43.3 kDa
protein
Codon usage
Phe
uuu
uuc
Leu
UUA
UUG
CUU
cue
lie
Met
Val
Ser
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
UCU
UCC
UCA
UCG
AGU
AGC
Pro
ecu
Thr
CCC
CCA
CCG
ACU
ACC
ACA
ACG
Trp
532
UGG
total content
22
6
23
13
11
2
4
7
22
10
5
13
7
4
6
8
4
9
7
9
2
5
3
5
1
5
9
6
6
12
28
60
codon usage
Ala
Tyr
His
37
13
Gin
Asn
Lys
25
Asp
Glu
31
Cys
Arg
14
26
12
Gly
total content
GCU
GCC
GCA
GCG
UAU
UAC
13
5
13
9
4
3
CAU
CAC
CAA
CAG
3
2
8
5
AAU
AAC
AAA
AAG
9
1
6
3
GAU
GAC
GAA
GAG
UGU
UGC
CGU
CGC
CGA
CGG
AGA
AGG
9
1
5
2
1
1
1
0
10
GGU
GGC
GGA
GGG
13
9
7
9
38
8
4
1
40
13
10
10
12
Nucleic Acids Research
from Tn10 and gives the araino acid composition for the 43.3 kDa
protein. The codon usage is clearly non-random and rather different from other E. coll proteins [19]. Very striking is the
use of UUU 22 times out of 28 codons for Phe. A similar observation has been made for the transposon Tn3 genes [20,21] as well
as others [21]. The general tendency seems to be that codons
with AU pairs instead of GC pairs in the wobble positions are
preferred in genes with a high rate of expression [21]. This
observation is especially clear in the cases of Phe, Leu, lie,
Ser, Ala, Gin, Asn, Asp, Glu, and Arg.
Distribution of amino acids in the tet resistance protein.
Figure 7 displays the distribution of amino acids grouped according to their polarity in hydrophobic, uncharged polar, acidic, and basic residues. The dominance of hydrophobic amino
acids, which make up for 58% of the total, is clearly demonstrated in this figure. With two exceptions they are evenly distributed over the entire protein. This result agrees well with the
proposed function of the resistance protein as an inner membrane
protein [22-24]. Two interruptions of the hydrophobic structure
occur at position 180-200 and 320-340. The former does not fall
in a region of horaology with pBR322 and may, therefore, not be
of functional importance. The latter, however, shows some homology with pbR322 suggesting a possible functional significance
(compare also fig. 8 ) .
Comparison of the tetracycline resistance genes in TniO and
pBR322. Figure 8 compares the amino acid sequences of the pBR322
encoded tetracycline resistance proteins [8] with that encoded
by Tn10. Clearly extensive homology exists between the two proteins encoded by pBR32 2 and the one encoded by Tn10. The homology is very good over the first 80 ainino acids of the small 21
kDa pBR322 protein and remains good throughout the next 80 amino
acids. The last 30 ainino acids of the small protein are not homologous to Tn10. Around position 160 of the small pBR322 tetracycline resistance protein the homology shifts to the large 40
kDa tetracycline resistance protein (compare fig. 8 ) . This is
coded in a different reading frame but is initiated within the
reading frame of the 21 kDa protein. It shows no horaology to the
Tn10 protein in that part which overlaps the 21 kDa protein to
533
w
Figure 7
H.t
Trt
Ala
Ser
TSr
Trr
*i"
6ln
Glu
Air
vai
H€t
I"
ft?
Hit
Asp
20
I
30
40
I
50
60
I
70
JllllL
80
90
L
m
JLL
J_
J
L
J
100 110 120 130 140 150 16O 170 180 190
ii i ii i
L
200
210 220 230 240 250 260 270 280 290 3OO 310 320 33O 340 350 360 370 380 390 400 410
L
10
L
i ill Hi I
D i s t r i b u t i o n of h y d r o p h o b i c amino a c i d s i n t h e 4 3 . 3 kDa p r o t e i n
Amino Acids
Amino Acids
Position of Ammo Acid
Acidic
Basic
Ala
f/?
vS?
Uncharged polar fH'
Ammo Acids
Hydrophobic
Ammo Acids
Nucleic Acids Research
position 160. Good homology with the Tn10 tetracycline resistance
protein is observed with the last 260 amino acids. It is interesting to note that two proteins, each of which shows extensive
homology to the respective Tn10 protein, are apparently necessary for tetracycline resistance in pBR322. This observation
will be discussed below in the light of TniO complementation
data [6].
DISCUSSION
The finding of a complex, overlapping promotor system for
the tetracycline resistance gene carried on Tn10 and described
in figure 3 is in agreement with previously published data [2].
A similar arrangement of overlapping promotors has been described for the tet proraotor of pBR322 [25]. The particular advantage of such an arrangement for the regulation of the tet
gene is not clear.
The transcription terminator structure is quite similar to
other terminators [16]. It is preceeded by translation termination codons in all three possible reading frames. Therefore, it
is very likely that this sequence is a functional terminator of
transcription. It remains unclear whether or not the possible
hairpin structure preceeding the terminator has any biological
significance. It displays the typical attenuator arrangement;
however, we did not find possible alternative structures typical
of attenuators [17].
The DNA sequence on the 1275 bp Hinc II fragment plus 165 nucleotides from the Hp_a I site on the 675 bp Hpa I - Hind III
fragment contains only one open reading frame which encodes a
protein of 43.3 kDa. Translation of this sequence terminates
downstream of the Hpa I site at position 1266. This agrees with
the observation that insertion of the 1275 bp DNA into a plasraid
leads to the synthesis of a fusion protein [1] and is consistent
with the mapping of mutants affecting tetracycline resistance
[26].
The molecular weight of 43.3 kDa, however, does not agree
with the apparent molecular weight of 36 kDa found in minicell
assays [1,2,5]. This discrepancy may be due to the high content
of hydrophobic amino acids in the protein which is known to reduce the apparent molecular weight on Laemmli gels in many cases
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Nucleic Acids Research
A
T
A
T
A
T
A
T
B
A
T
B
A
T
B
A
T
B
A
T
B
A
T
B
Met Lys Ser Asn Asn
Met Asn Ser
Asp
Leu
Ala Val Glv
Leu Asp Ala Met Gly
Leu Leu Arq Asp H e
Leu Leu Arq Glu Phe
Glv Val Leu Leu Ala
Glv Val Leu Leu Ala
Ala Leu H e Val
Ser Thr Lys H e
H e Glv Leu, Val
H e Gly Leu H e
Val His Ser Asp
Tie Ala Sen r,iu
Leu Tvr Ala Leu
Leu Tvr Ala Leu
Pro Val Leu G.1Y Ala Leu Ser
Pro Trp Leu Glv Lys Met Ser
Thr Arg Ser Arg Ser Thr Val
Leu Leu Ala Ser Leu Leu Gly
Leu Leu Leu S«r Leu H e Glv
Pro Ala Arg Phe Ala Thr Trp
Ala Thr Thr Pro Val Leu Trp
Ala Phe Ser Ser Ala Leu Trp
Gly Asp His Thr Arg Pro Val
He
Ala
Met
Met
Ser
Asp
Met
Met
Ara Phe Glv Arq Arq
Arq Phe Glv Arq Arq
Arg Pro Leu Trp Pro Pro
Ala Thr He Asp Tvr Ala
Ala Ser Leu A S P Tvr Leu
Ser His Tyr Arg Leu Arg
He Leu Tvr Ala Gly Arq
Met Leu Tvr Leu Gly Arq
Asp Pro Leu Arg Arg Thr
ASP
ASP
A
T
B
Ala Glv lie Thr Glv Ala Thr Glv Ala Val
Ser Glv lie Thr Glv Ala Thr Glv Ala Val
Gly Arg His His Arg Arg His Arg Cys Gly
Ala A S D H e Thr Asp Gly Glu Asp Ara Ala
Ala A S P Thr Thr Ser Ala Ser Gin Arq Val
Arg Arg His His Arg Trp Gly Arg Ser Gly
Met Ser Ala Cvs Phe Gly Val GlY Met Val
Leu Glv Ala Ser Phe GlY Leu Glv Leu H e
His Glu Arg Leu Phe Arg Arg Gly Tyr Gly
Glv Asp Cys Trp Ala Pro Ser Pro Cys Met
Glv GlY Phe Ala GlY Glu He Ser Pro His
Glv GIY Leu Leu GlY Ala H e Ser Leu His
Arg Arg Cys Met Thr Ala Ser Thr Tyr Tyr
Ala Ala Leu Leu Asn Tip Val _Thr Phe Leu
Ala Ala Val Leu Asn Gly Leu Asn Leu Leu
T
B
T
B
T
B
T
B
Phe
Met
Gly
Ala
Thr
He
Glv
Gly
Arq
Gin
Val
Phe
Met
Val
Gin
Gin
Glu
Glu
Glu
Asn
Pro
Ala
He
Val
T
B
T
B
T
Phe
Phe
Gly
Gly
Ala
Thr
Gly
Arq
Leu
He
Thr
Lys
Trp
Trp
Leu
Leu
Lys
Arg
A
T
B
A
T
B
3
536
Thr
Ser
Thr
Pro
He
Ala
Pro
Pro
Lys
His
Gin
Val
T.en
Leu
Ala
Ala
Asn
Lys
Ser
Ser
Leu
Met
Thr
Ala
Asn Ser Met
Ser Ala Thr
His Ser Val
His Ala Leu
Trp Gly Glu
Phe Glv Glu
Thr
Gly
Asn
Ser
He
Thr
Val
Leu
Met
Met
Phe
Ala
Lys
Lys
Leu Gly Thr Val Thr
Leu Val H e Thr Leu
Pro Ye* Leu Pro ,Gly
Pro Va.1, Leu Pro Thr
H e Ala Ser HJs Tvr
Ile- Ala Asft H i * Phe
Gln Phe Leu Cys Ala
Gin Val H e Phe Ala
Met Arg
Arg
Glu
Ser
Phe
He
Val
Trp
Trp
Val
H e
Gin
Gin
Thr
Gin
Asp
Arg
Val
Arg
Tyr
Phe
Val
Val
Glv
Glv
Ala
Ala
Ala
Ala
Pro Val
Pro Val
Ser
H e Met
Leu Leu
Asp His
He Val
Leu Leu
His Arq
Ala Gly Ala Tyr He
Ala Ala Ser Val H e
Cys Trp Arq Leu Tyr
Arg His Phe Glv. Leu
Lys Trc Phe Glv Trp
Ser Pro Leu Arg Ala
Ala QlY Pro Trp Pro
Ala GlY Pro . H e H e
Gly Arg Pro .Val Ala
His His Ser Leu Arg
Ser Pro Phe Phe He
Ala Pro Phe Leu Ala
Trp Ala Ala Ser term
Val Val Met Phe Trp
Leu Gly Cys Phe Leu
Asn Thr Asp Thr Glu Val
Arg Pro Met Pro Leu Arq
Tyr H e Thr Leu Phe Lys
Trp Ala Arg Gly Met Thr
Phe. Ser Ala G3n Leu H e
Phe H e Met Gin Leu Val
Leu Phe Thr Glu Asn Arq
H e Phe Gly Gin Asp Arq
Phe Ser Leu Ala Gly Leu
Leu Ser Leu Ala Val Phe
Phe Val Ala Gly Arq H e
Phe Val Thr Glv Pro Ala
Val Leu Leu Glu Phe H e
H e H e Ala Gly Met Ala
Nucleic Acids Research
T
B
T
B
T
B
T
B
T
B
T
B
T
B
T
B
Ala Asp Ser Ser
Ala Asp Ala Leu
Trp Leu Asp Phe
Trp Met Ala Phe
Ala Leu Pro Ala
Gly Met Pro Ala
His
Asp
Ala
Leu
His
Ala
Ala
Ala
Glu
His
Thr
Thr
Ser
Ser
Phe
Leu
Gin
Gin
Gly
Ser
Leu
Ala
Tyr
Tyr
Ala
Gly
Pro
Pro
Leu
Leu
Phe
Tyr
Val
He
Ala Phe Leu
Val Leu Leu
Leu H e Leu
Met H e Leu
Gin Gly Val Met
Gin Ala Met Leu
Gly Ala Leu
Gly Gin Leu
Val H e Gly
H e Thr Gly
Pro H e Trp
Ser Thr Trp
Gin
Gin
Pro
Pro
Asp
Asn
Cys lie He H e
Leu Val Cys Leu
Thr Pro Gin Ala Gin Gly Ser
Ser Arg Ala Thr Ser Thr term
Gly
Gly
Leu
Leu
Gly
Gly
Leu
Pro
Lys
Leu
Ser
Leu
He
Trp
Leu
Leu
Ala
Gin
Ala
Ala
Leu
Leu
Ser
Ser
Phe H e Ser Glu Gly
Phe Ala Thr Arq Gly
Ala
Ala
He
Arq
Gly Gly Gly H e
Ser Giy Gly H e
Gin Thr Lys Ser
Gin Val Asp Asp
Val
Ala
Thr
Thr
H e Trp
Ala Trp
Ser Leu Thr Asn
Ala Leu Thr Ser
Leu
Leu
Phe
Val
Val H e Tyr
Ala H e Tyr
H e H e Gly
H e Val Gly
Ser Met Thr Phe Met
Leu Arg Arg Gly Ala
Asn
Ala
Leu
Ala
Leu
Trp
Glu Thr Ser Ala term
Figure 8 Comparison of the amino acids
sequences
of the
Tn10(A)
encoded 4 3.3 kDa protein (T) with the pBR322
encoded
21 kDa
and 40 kDa (B) tetracycline resistance proteins [8] . Homologous
amino acids are underlined.
[27], Indeed, using a phosphate buffered SDS polyacrylaraide gel
system, a molecular weight of 50 kDa has been found for the TET
protein instead of 36 kDa as seen in the glycine buffered gel
system [5], Therefore, we conclude that the real molecular
weight of the TET protein is 4 3.3 kDa. However, it may also be
processed resulting in a reduced molecular weight.
The existence of two complementation groups for tet resistance has led to the conclusion that there are two genes being
carried on this DNA [6]. Complementation may also occur, however,
at the protein level, if it is assumed that an oligomer of the
4 3.3 kDa protein is the functional unit. This is supported by the
situation of the tet genes on pBR322 where two proteins are encoded in the tet resistance sequence [8]. The existence of homology between pBR322 TET proteins and the Tn10 TET protein favours this interpretation. Interestingly, homology does not occur
between the amino terminal end of the second pBR322 protein,
which overlaps with the first gene, and the TET protein from
Tn10. Taken together, the existence of two functional domains
on the TET proteins seems to be possible and may explain the
complementation results [6].
Although hybridisation experiments revealed that pBR322 and
537
Nucleic Acids Research
Tn10 fall into different classes with respect to their tetracycline resistance determinants due to a lack of DNA homology
[7], the amino acid sequences of the resistance proteins show
good homology. This apparent contradiction can be explained by
differences in the use of wobble bases.
The gene start for the 17.6 kDa protein in the same reading
frame as the 4 3.3 kDa protein shows a good Shine-Dalgarno sequence [18]. This may be
the low molecular weight tetracycline-
inducible protein which has been found in other experiments [5].
Within the DNA sequence reported here it is the only possible
open reading frame for a protein in the molecular weight range
of 15 kDa [5].
ACKNOWLEDGEMENTS
We wish to thank Dr. R, Schmitt and Dr, H C G. Gassen for many
helpful discussions, Dr. S..H. Waters for sharing his unpublished
results with us, M. Luxnat and I t Kaffenberger for their assistance in preparing some of the DNAs for sequencing, and E.
R6nnfeldt for typing the manuscript. This work was supported by
a grant from the Chemische Fabrik RiJhm GmbH, Darmstadt.
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