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Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(6):
621-628
CN 31-1300/Q |
A
Negative Element Located in the Upstream Flanking Region of the Gene Encoding
Arginyl-tRNA Synthetase (argS) from Escherichia coli
( State Key Laboratory of Molecular
Biology, Institute of Biochemistry and Cell Biolog,
ArgRS
with glutamyl-tRNA and glutaminyl-tRNA synthetases differs from the other 17
aaRSs in that it requires the cognate tRNA to catalyze the ATP-PPi exchange
reaction[12]. ArgRS from E. coli has been studied in our
laboratory[12-15]. The gene, argS, encoding ArgRS was cloned
as a fragment of 2.4 kb[11]. It contains an open reading frame of 1
734 nt, and 5′ and 3′flanking regions of 247 nt and 397 nt. ArgRS contains 577
amino acid residues with a molecular weight of 64.8 kD. In the E.coli
TG1 transformant containing the recombinant plasmid of pUC18 and argS (named
pUC18-argS), ArgRS was overproduced 1 000 fold as compared with that in
the host cells[16]. Leucyl-tRNA synthetase (LeuRS) from E.coli
has been another focus of study in our laboratory[17-22]. Its gene leuS
with 5′ and 3′flanking regions was cloned and over-expressed also. However, in
the E.coli TG1 transformant containing the recombinant plasmid of pUC18
and leuS, LeuRS was only overproduced 35 fold as compared with that in
the host cells[20]. The overproduction of ArgRS seems not to be
caused by the high copy number of pUC18. Why was argS expressed so much?
What is the effect of the 5′flanking region on the expression of this gene? In
the present paper, by a series of deletion mutations in the 5′flanking region
of argS, a 19 nt negative element in this region was detected, its
action on transcription of argS was studied and the effect of arginine
on the gene expression was reported.
1.1
Materials
A
RNA isolation kit was purchased from Watson Biotechnology Inc. The kits for non-radioactive
(digoxigenin) labeling and detection of mRNA were obtained from Boehringer
Mannherm. The [a-32P] dATP was the product of Amersham (England).
The plasmids pUC18 and pSP72 were purchased from Promega Co.. The primers for
deletion mutations were synthesized by a DNA synthesizer at the Shanghai
Institute of Plant Physiology, the Chinese Academy of Science. The T7 and SP6
primers for preparation of DIG-labeled RNA probe were obtained from Sangon Co..
The total tRNA containing more than 70% of tRNA2Arg was
isolated from an overproducing E. coli strain containing tRNA2Arg
gene in our laboratory[23]. The T7 RNA polymerase was purified by
the method of Li et al[24].
1.2
Methods
1.2.1 Construction
of directional deletion The
deletion mutation was carried out by PCR. Synthetic oligonucleotides DL1 (TAA GGA
TCC GTG AAT ATT CAG GCT CTT CTC), DL2 (TAA GGA TCC TAA GGT ATT CCG
GTG AAT ATT), DL3 (TAA GGA TCC CGC CCT AAT TTC TTT AAC), DL4 (TAA GGA
TCC TAT ACT CCC GCC CTA ATT TC), DL5 (TAA GGA TCC TGA CGA AAA CAG
CCA TTT G), DL6 (TAA GGA TCC CGC CAC GCG CAC), DL7 (TAA GGA TCC
AAT AGT CAA AAC GGC AAT ACG) and DL8 (TAA GGA TCC CTG TTG CCC TGT GCCA)
with BamHI site (underlined) at 5' end, and primer SP (GCC AAG CTT CAC
CAT AGG CTT, its 5' is complementary with the sequence of 3' end of argS)
with HindIII site (underlined) at 3' end, were used as the primers.
Using argS as a template, the deleted mutants in 5' flanking regions of argS
with various lengths were amplified (Fig.1). The mutants were digested by BamHI
and HindIII and inserted into the gap of plasmid pUC18 treated with the
same enzymes to form the recombined plasmid that was termed as pUC18-argSDn
(Fig.1). The DNA sequences of the deletion mutants were confirmed by dideoxy
sequencing.
argSD1,
deletion from -189 to +60; argSD2, deletion from -189 to +48; argSD3,
deletion from -189 to -7; argSD4, deletion from -189 to -12; argSD5,
deletion from -189 to -34; argSD6, deletion from -189 to -52; argSD7,
deletion from -189 to -70; argSD8, deletion from -189 to -122.
1.2.3 Assays
of aminoacylation activity The
aminoacylation activity of ArgRS was measured as previously described[12].
The assays were performed at 37 ℃
in a reaction mixture of 25 ml which contained 50 mmol/L Tris-HCl, pH 7.4, 8
mmol/L MgCl2, 4 mmol/L ATP, 80 mmol/L KCl, 0.5 mmol/L dithiothreitol
(DTT), 0.1 mmol/L EDTA, 20.5 g/L total tRNA (corresponding to 534 mmol/L of
tRNA2Arg) and 0.1 mmol/L [3H]arginine (25
mmol). One unit was defined as the amount of enzyme that charges 1 nmol
arginine to tRNAArg in 1 min under the standard condition. The
specific activity was defined as the units of enzyme per milligram protein. The
Km values for the three substrates were determined under
similar condition, except that the concentrations of the corresponding
substrates were varied. The concentration of purified ArgRS was determined by A280
of the enzyme solution. About 1.18 g/L of protein equaled 1 A at 280 nm.
1.2.4 Purification
of the enzyme from both inclusion bodies and extract supernatants The transformants
carrying the recombinant plasmid pUC18-argS or pUC18-argSDn were
grown in 500 ml of LB containing 50 mg of ampicillin at 37 ℃
to a stationary phase. Cells were harvested by centrifugation,
then suspended in 40 ml of disruption buffer (pH 7.5, 100 mmol/L of Tris-HCl,
10 mmol/L of MgCl2, 1 mmol/L of EDTA) and sonicated for 15×30
seconds at 15 watts with a High Intensity Ultrasonic Processor (375-watt
model). After centrifugation at 12 000 r/min, 4 ℃
for 30 mins, the supernatant and precipitate were separately fractionated by
SDS-PAGE to detect their amounts of ArgRS. If an inclusion body was formed
during expression of ArgRS, the purification of the enzyme was performed by
denaturation of the inclusion body and renaturation of dilution[25].
Purification of ArgRS in the supernatant was carried out by a two-step
chromatography on DEAE-Sepharose CL-6B and Blue Sepharose columns, according to
the previous method in our laboratory[26].
1.2.5 Isolation
of total cellular RNA The
total RNA was extracted from exponential-phase cultures of TG1 transformants
grown in LB or M9 by mixing the cells with Trizol reagent in the RNA isolation
kit purchased from Watson Biotechnology Inc. The RNA concentration was
determined by the A260 of the RNA solution (20 A260
equal to 1 mg of RNA).
1.2.6 Preparation
of DIG-labeled RNA as probe There
is one site of ClaI and SalI in the coding region of argS,
respectively. About a 1 kb DNA fragment of argS digested with the above
two enzymes was inserted into the gap of transcriptional vector pSP72 cut with
the same enzymes. Using the recombinant plasmid as a template and T7 and SP6
primers by PCR a DNA fragment was amplified and used as a template for in
vitro transcription of the DIG-labeled probe. The RNA probe was synthesized
and labeled by T7 RNA polymerase and digoxigenin-11-UTP in an in vitro
transcription reaction using a DIG Northern blot starter kit from Boehringer
Mannheim.
1.2.7 RNA
Northern dot hybridization The
total RNA prepared from the TG1 transformant carrying the recombinant plasmid
pUC18-argS or pUC18-argSDn was denatured by the method of Thomas[27].
The denatured RNA was dotted on a positively charged nylon membrane (Boehringer
Mannheim), fixed by UV-crosslinking with an UV-cross linker, hybridized with
DIG-labeled RNA probe, and detected by anti-digoxigenin-AP (alkaline
phosphatase).
1.2.8 Gel
retardation analysis
Gel
retardation analysis was carried out as described by Champagne et al.[10].
In the reconbinant plasmid, EcoRI is located in the multicloned site of
pUC18 and in front of argS, the AccI cutting site is at -11 nt of
argS. The EcoRI-AccI fragments, obtained from pUC18-argSD7
and pUC18-argSD6, were labeled at their extremities by Klenow enzyme and
a-32P]dATP. The extract from the TG1 strain was used to supply the
putative trans-regulation factor. Approximately 0.1 pmol of the
end-labeled EcoRI-AccI fragments were incubated for 20 min at
room temperature with different volume of the extract. Factor-DNA complexes
were prepared in 20 ml of 100 mmol/L Tris-HCl, pH 7.5 containing 30 mmol/L
NaCl, 2 mmol/L EDTA, and 1 mmol/L dithiothreitol. 9 mg/L of Poly (dI·dC)
(Pharmacia) was added to inhibit nonspecific binding of protein to the DNA. The
reaction mixtures were separated by electrophoresis on a 12% polyacrylamide gel
in 0.5×TBE buffer at room temperature and visualized using autoradiography.
1.2.9 Analysis
of the secondary structure of mRNAs of argS and leuS Energy of the secondary structure around the Shine-Dalgarno
sequences of mRNAs of argS and leuS was measured by Michael
Zuker's MFold program of WPI (Wisconsin Sequence Analysis Package).
2.1 Effect
of the deletion in the 5′flanking region on the expression of argS
The
total protein in the whole cells containing argS and argSDn was
extracted and denatured with boiling loading buffer of SDS-PAGE and then
fractionated by SDS-PAGE. The strong band of ArgRS with a molecular weight of
64.8 kD appeared in the extracts from the cells containing argSD8 (the
upstream of -122 nt was deleted), argSD7 (the upstream of -70 nt was
deleted), argSD6 (the upstream of -52 nt was deleted), argSD5
(the upstream of the -35 region was deleted) and argSD4 (the upstream of
the -10 region was deleted). The above argSDns were still over-expressed
almost as high as argS. But no band of ArgRS appeared in the extracts
from the cells containing argSD3 (-10 region was deleted), argSD2
(SD region was deleted), argSD1 (without 5′flanking region) (Fig.2 and
Table 1).
Molecular weight markers from Sigma, with
molecular weight of 97.4, 66.0, 45.0, 31.0, 21.5 and 14.5 kD (lane 1); purified
ArgRS (lane 2); proteins in the crude extract of TG1 transformants containing argS
(lane 3), argSD8 (lane 4), argSD7 (lane 5), argSD6 (lane
6), argSD5 (lane 7), argSD4 (lane 8), argSD3(lane 9), argSD2
(lane 10), argSD1 (lane 11); proteins in the crude extract of TG1 (lane
12). In each lane 15 ml of sample was loaded.
2.2 A
negative element of A, T-rich sequence of 19 nucleotides located between –70 nt
and –52 nt of argS
After
disruption of transformants containing argSDn by sonication and separation
of the supernatant and precipitate by centrifugation, the activity of
aminoacylation was determined. In the supernatant from argSD4, argSD5
and argSD6, the aminoacylation activity was only about 60% of the
activity of that of the transformant containing argS. However, the
result of SDS-PAGE showed that the total amount of ArgRS in the whole cells of
the above different transformants was similar (Fig.2). When assay of the amount
of ArgRS in precipitate and supernatant from the transformants containing argS
and argSDn by SDS-PAGE, the band of ArgRS appeared both in the
precipitate and supernatant from those containing argSD4, argSD5,
and argSD6, respectively. A strong band of ArgRS appeared only in the
supernatant from the transformants containing argS, argSD7 and argSD8,
however ArgRS was detected both in the supernatant and precipitate from those
containing argSD4, argSD5 and argSD6 (Fig.3). An inclusion
body of ArgRS was formed partially during the expression of argSD4, argSD5
and argSD6 in those the upstream of –52 nt was deleted. During the
expression of wild type argS, or argSD7 and argSD8 in
which the nucleotide fragment between –70 and –52 nt was kept, the formation of
an inclusion body was not detected.
Molecular weight markers from Sigma, with
molecular weights of 97.4, 66.0, 45.0, 31.0, 21.5 and 14.5 kD (lane 1);
purified ArgRS (lane 2); proteins in the crude extract of TG1 transformants
containing argSD6 (lane 3), argS (lane 4), argSD5 (lane
5), argSD4 (lane 6); proteins in the supernatant of crude extract of TG1
transformants containing argSD6 (lane 7), argS (lane 8), argSD5
(lane 9), argSD4 (lane 10); proteins in the precipitant of crude extract
of TG1 transformants containing argSD6 (lane 11), argS (lane 12),
argSD5 (lane 13), argSD4 (lane 14). In each lane 15 ml of sample
was loaded.
Northern
hybridization experiments were carried out to identify the effect of argSDn
on transcription of argS. The total RNA was isolated from transformants
contained argS, argSD4, argSD5, argSD6 and argSD7.
Different amounts (serial dilutions) of these RNAs were applied to a sheet of
positively charged nylon membrane and hybridized with the DIG-labeled RNA
probe. The levels of the specific mRNA encoding ArgRS from transformants
contained argSD4, argSD5 and argSD6 were about 2-3
times than those of argS and argSD7 (Fig.4). When the nucleotide
between -70 nt and -52 nt in argSD4, argSD5 and argSD6 was
deleted, the amount of mRNA was increased. The result showed that the
expression of argS during transcription of argS declined by the
negative element.
Total RNA was isolated from TG1(pUC18-argSD7)
(lane 1), TG1(pUC18-argS) (lane 2), TG1(pUC18-argSD4) (lane 3),
TG1(pUC18-argSD5) (lane 4), TG1(pUC18-argSD6) (lane 5),
respectively. The cells were cultured in LB to exponential-phase. Different
amounts of RNA were used in all samples. From top to bottom the amount of
sample were 0.5, 1.0 and 2.0 mg, respectively. The lower panel shows
integration done by use of Shimadzu spectrodensitomery of the dot blots.
When
the transformant containing argS was cultured in minimal medium or supplemented
with the same concentrations of Arg (cognate amino acid of ArgRS), Ala (small
amino acid without charge) and Lys (with positive charge), separately, or the
other 19 amino acids except Arg, the band of ArgRS appeared on the gel of PAGE
only in the transformant cultured in the medium supplemented with Arg
[Fig.5(A)]. The same case was found in the transformant containing argSD7
or argSD8 (data not shown). But to the transformant TG1 (pUC18-argSD4)
or TG1 (pUC18-argSD5) or TG1 (pUC18-argSD6), the band of ArgRS
was present in all cases (data not shown). The total RNAs from the
transformants TG1 (pUC18-argS) or TG1 (pUC18-argSDn), grown in
minimal medium or M9 medium supplemented with 10 mmol/L Arg or Lys or other
amino acid except Arg, was analyzed by Northern dot blot hybridization. Arg
specially induced the transcription of argS bringing the negative
region, however it did not affect the transcription of argS without the
negative region [Fig.5(B)].
(A) SDS-PAGE of total protein in
transformants TG1(pUC18-argS) cultured in minimal medium unsupplemented
or supplemented with some amino acid. Molecular weight markers from Sigma, with
molecular weights of 97.4, 66.0, 45.0, 31.0, 21.5 and 14.5 kD (lane 1);
purified ArgRS (lane 2); proteins in the crude extract of transformants
TG1(pUC18-argS) cultured in minimal medium (lane 3), or added 2 mmol/L
alanine (lane 4), 10 mmol/L alanine (lane 5), 2 mmol/L lysine (lane 6), 10
mmol/L lysine (lane 7), 2 mmol/L 19 other amino acids except arginine (lane 8),
10 mmol/L 19 other amino acids except arginine (lane 9), 2 mmol/L arginine
(lane 10), 10 mmol/L arginine (lane 11). In each lane 15 ml of sample was
loaded.
(B) Dot blot hybridization of total RNA
from transformants TG1(pUC18-argS) or TG1(pUC18-argSΔ5) cultured
in minimal medium unsupplemented or supplemented with some amino acid with the argS
internal probe. The transformants TG1(pUC18-argS) were grown in minimal
medium (lane 1), or supplemented with 10 mmol/L Lys (lane 2), or supplemented
with 10 mmol/L Arg (lane 3). The transformants TG1(pUC18-argSD5) were
cultured in minimal medium (lane 4), or supplemented with 10 mmol/L Lys (lane
5), or supplemented with 10 mmol/L Arg (lane 6). Different amounts of RNA were
used in all samples. From top to bottom the amount of sample were 1.0, 2.0 and
3.0 mg, respectively.
To
examine whether there was a factor to participate in argS expression, an
in vitro interaction of a factor with the putative negative region
located 5′flanking region of argS was studied by gel retardation
technique. The results in Fig.6 showed that the EcoRI-AccI DNA
fragment from argSD7 labeled with [a-32P]dATP was retarded,
however, the fragment from argSD6 was not. This suggested that a factor
might bind on the negative element, so that the labeled band was retarded. The
amount of the retarded band related directly with the amount of the extract
from E.coli TG1, and the more extract, the stronger retardation.
The fragments of pUC18-argSD7 and
pUC18-argSD6 extended from EcoRI site to AccI site,
corresponding to the fragment from -70 nt and -52 nt to -11 nt of argS,
respectively. The fragments from argSD7 (lane 1-4) and argSD6 (lane 5-8)
were mixed with 0, 2, 5, and 10 ml of extract from TG1, respectively. The
arrowhead points the band retardation.
The
result of the deletion mutations of the 5′flanking region of argS showed
that the full-length of 5′flanking region was not necessary to expression of
ArgRS. In E.coli TG1 transformant containing argS and some argSDn,
ArgRS can be overexpressed with similar amount (Fig.2). The –10 region (TATA
box) of promoter is necessary for the expression. With further deletions of the
flanking region downstream of TATA box, ArgRS was not produced, suggesting that
overexpression of argS was not due to the multi-copy number of pUC18 and
argS might have an own strong promoter. The level of transcription of
the gene was higher and a part of the enzyme formed an inclusion body, when the
upstream of –52, –35 or –10 nt of argS was deleted (Fig.3 and Fig.4).
However, no inclusion body was formed and its transcription level was similar
to that of wild type argS when the upstream of –70 nt was deleted (Fig.3
and Fig.4). The above results suggested that in argS a 19 nt A, T-rich
special sequence located between –70 and –52 nt regulated the normal
transcription of argS. It was a negative element, and its deletion
resulted in faster expression of ArgRS and formation of an inclusion body. Gel
retardation analysis also showed that a factor might bind to the negative
element and regulate the expression of the gene.
The
result of SDS-PAGE indicated expression of argS in E.coli was
specifically induced by arginine [Fig.5(A)]. And the result of Northern dot
blot hybridization [Fig.5(B)] showed that it functioned at the level of
transcription, which was also found in Brevibacterium lactofermentum (B.
lactofer-mentum) argS[28]. The situation is very
different from that was found in many other genes coding for aaRSs in E.coli
and B.subtilis. Expression of these genes is induced by starvation for
cognate amino acid and not by general amino acid limitation[1].
However, regulation of expression of argS in B. subtilis still
has not been reported yet.
A
key feature of genetic regulatory system is the ability to target the system to
specific genes in response to specific physiological signals. The effect of
arginine might be related with the negative element. Arginine might repress the
binding of the factor to the negative element to provide enough ArgRS to
decrease the cellular concentration of arginine. However, in the biosynthetic
operon of arginine, the excessive end product arginine, in conjunction with the
arginine repressor protein, represses the synthesis of the 10 enzymes of
arginine biosynthesis[28]. Because E. coli is not able to use
arginine as an efficient carbon and nitrogen source, excessive arginine might
be harmful to the cell, maybe via to disturbing the pH in the cells[28].
Although the effect of arginine on argS and arginine biosynthetic operon
is opposite, the ultimate aim is to reduce the cellular concentration of
arginine.
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Received: June 4, 2001 Accepted:
June 27, 2001
This work was supported by Grant No.
39670412 from the National Natural Science Foundation of China
*Corresponding author: Tel,
86-21-64374430; Fax, 86-21-64338357; e-mail, [email protected]