| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 34, 32300-32306, August 22, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
Received for publication, May 7, 2003 , and in revised form, June 10, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The mazEF system, which consists of two adjacent genes, mazE and mazF, is located downstream from the relA gene on the E. coli chromosome. Sequence analysis revealed that they are partly homologous to the pemI and pemK genes on plasmid pR100 (23). The mazEF system has the properties required for an addiction module. MazF is toxic and MazE is antitoxic; MazF is stable, whereas MazE is a labile protein degraded in vivo by the ATP-dependent ClpPA serine protease (22); MazE and MazF are coexpressed and interact with each other to form a complex; and the expression of mazEF is negatively autoregulated by MazE and the MazE-MazF complex (24). The expression of mazEF is also inhibited by guanosine 3',5'-bispyrophosphate (ppGpp),1 which is synthesized by the RelA protein under extreme amino acid starvation (22). The mazEF-mediated cell death can be triggered by extreme amino acid starvation and thymine starvation (25), by toxic protein Doc (26), and by some antibiotics that are general inhibitors of transcription and/or translation, such as rifampicin, choramphenicol, and spectinomycin (27).
Some crucial aspects of the mazEF system have remained elusive. It is still unknown how MazE or the MazE-MazF complex binds to the mazEF promoter DNA and how MazE and MazF interact with each other to form a complex. In the present paper, we have investigated the interactions between MazE, MazF, and the mazEF promoter DNA to identify the functional domains in MazE responsible for binding to the mazEF promoter DNA and for interacting with MazF. It is demonstrated that MazE has a DNA-binding domain in its N-terminal region and that the region from residues 38 to 75 in MazE is required for its binding to MazF, in which Leu55 and Leu58 are essential. The data in the present paper also suggest that the MazE-MazF complex consists of one MazE dimer and two MazF dimers.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Constructions of Plasmid—The mazEF gene (including
its Shine-Dalgarno sequence region) was amplified by PCR using E.
coli genomic DNA as template and cloned into the
XbaI-NheI sites of pET11a, creating the plasmid pET11a-EF.
The mazEF gene (including its Shine-Dalgarno sequence region) was
amplified by PCR and cloned into the XbaI-XhoI sites of
pET21cc to create an in-frame translation with a (His)6 tag at the
MazF C terminus. The plasmid was designated as pET21cc-EF(His)6.
The mazE gene was amplified by PCR and cloned into the
NdeI-HindIII sites of pET28a. This plasmid was designated as
pET28a-(His)6E. MazE was expressed as a fusion with an N-terminal
(His)6 tag followed by a thrombin cleavage site, named
(His)6MazE. The full-length mazE gene and various
N-terminal and C-terminal deletion constructs of the mazE gene as
indicated in Fig. 7 were
generated by PCR and cloned into EcoRI-PstI sites of pGAD-C1
vector to create in-frame translation fusions with the Gal4 transcriptional
activation domain. These plasmids were designated pGAD-MazE,
pGAD-MazE
(1–13), pGAD-MazE(1–24),
pGAD-MazE(1–37), pGAD-MazE(1–46),
pGAD-MazE(68–82), and pGAD-MazE(76–82). The
full-length mazF gene and various N-terminal and C-terminal deletion
constructs of the mazF gene were generated by PCR and cloned into
EcoRI-BglII sites of pGBD-C1 vector to create in-frame
translation fusions with the Gal4 DNA-binding domain. These plasmids were
designated as pGBD-MazF, pGBD-MazF(1–14),
pGBD-MazF(1–25), pGBD-MazF(72–111), and
pGBD-MazF(97–111).
|
Protein Purification—pET11a-EF was introduced into E.
coli BL21(DE3) strain. The coexpression of MazE and MazF was induced for
4 h in the presence of 1 mM isopropyl-
-thiogalactopyranoside.
The cells were harvested by centrifugation and lysed using French press. The
cell lysis was kept at 37 °C for 30 min to degrade MazE as much as
possible, and cell debris and unbroken cells were then removed by
centrifugation at 8,000 x g for 10 min followed by
ultracentrifugation at 10,000 x g for 1 h to remove membrane
and insoluble fractions. MazF was subsequently purified by ammonium sulfate
fractionation, gel filtration on Sephadex G-100 column, DEAE-Sepharose, and
hydroxyapatite column chromatography. The fractions containing MazF protein
were pooled and concentrated. MazF was further purified by gel filtration with
a SuperduxTM 200 column (Amersham Biosciences). For purification of
(His)6MazE, pET28a-(His)6E was introduced into E.
coli BL21(DE3) strain, and (His)6MazE expression was induced
with 1 mM isopropyl--thiogalactopyranoside for 4 h.
(His)6MazE protein was immediately purified by
nickel-nitrilotriacetic acid (Qiagen) affinity chromatography.
pET21cc-EF(His)6 was also introduced into E. coli
BL21(DE3) strain. The coexpression of MazE and MazF(His)6 was
induced in the presence of 1 mM
isopropyl--thiogalactopyranoside for 4 h. The MazE-MazF(His)6
complex was immediately purified by nickel-nitrilotriacetic acid (Qiagen)
affinity chromatography and further purified by gel filtration. To purify
MazF(His)6 from the purified MazE-MazF(His)6 complex,
MazE in the purified MazE-MazF(His)6 complex was dissociated from
MazF(His)6 in 5 M guanidine HCl. MazF(His)6
was retrapped by nickel-nitrilotriacetic acid resin (Qiagen) and refolded by
stepwise dialysis. The yield of refolding is 
80%. The biochemical
activity of MazF(His)6 was determined with E. coli T7 S30
extract system (Promega) for the protein synthesis inhibition.
Electrophoretic Mobility Shift Assays (EMSA)—Two
single-stranded oligonucleotides
(5'-GCTCGTATCTACAATGTAGATTGATATATACTGTATCTACATATGATAGC-3' and
3'-CGAGCATAGATGTTACATCTAACTATATATGACATAGATGTATACTATCG-5') were
synthesized and annealed to get the 50-bp double-stranded DNA containing the
mazEF promoter sequence. The 50-bp DNA fragment was end-labeled by T4
polynucleotide kinase with [
-32P]ATP and used to detect the
protein-DNA binding by EMSA. The binding reactions (20 µl) were carried out
at 4 °C for 30 min with purified proteins, 2 µl of 100 µg/ml
poly(dI-dC) and 2 µl of labeled DNA fragment in the binding buffer (50
mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1
mM dithiothreitol, and 5% glycerol). Electrophoresis was performed
in TAE buffer at 100 V in 6% native polyacrylamide gel. After electrophoresis,
the gel was dried and then exposed to x-ray film.
Native PAGE—Different amounts of (His)6MazE and
MazF were mixed in binding buffer (50 mM Tris-HCl, pH 7.5, 5
mM MgCl2, 1 mM dithiothreitol, and 5%
glycerol) at 4 °C for 30 min, and then 2x loading solution (40
mM Tris-HCl, pH 7.5, 80 mM -mercaptoethanol, 0.08%
bromphenol blue, and 8% glycerol) was added to the mixtures before loading on
a native gel. The composition of the stacking gel was 5% acrylamide-bis (29:1)
in 62.5 mM Tris-HCl, pH 7.5, and the composition of the separation
gel was 10% acrylamide-bis (29:1) in 187.5 mM Tris-HCl, pH 8.9. The
running buffer contains 82.6 mM Tris-HCl (pH 9.4) and 33
mM glycine. Electrophoresis was performed at constant voltage (150
V) at 4 °C. The protein bands were visualized by Coomassie Brilliant
Blue.
Resolution of Low Molecular Mass Proteins by Tricine SDS-PAGE— Tricine SDS-PAGE was carried out according to the method described previously (28) with some modifications as follows: stacking gel, 5% acrylamide-bis (48:1.5) in 0.75 M Tris-HCl, pH 8.45, and 0.075% SDS; spacer gel, 10% acrylamide-bis (48:1.5) in 1.0 M Tris-HCl, pH 8.45, and 0.1% SDS; resolving gel: 16.5% acrylamide-bis (48:1.5) in 1.0 M Tris-HCl, pH 8.45, and 0.1% SDS. The anode running buffer was 0.2 M Tris-HCl, pH 8.9, and the cathode running buffer was 0.1 M Tris base, 0.1 M Tricine, and 0.1% SDS. After running the gel at constant current (20 mAmp) at room temperature, the protein bands were visualized by Coomassie Brilliant Blue.
Assays of MazE-MazF Interaction in the Yeast Two-hybrid System— The yeast two-hybrid reporter strain PJ69–4A (MATa trp1-901 leu23,112 ura3-52 his3-200 gal4 gal80LYS2:GAL1-HIS3 GAL2-ADE2 met: GAL7-lacZ) and vectors pGAD-C1 and pGBD-C1 were used for two-hybrid assays (29). To localize the MazF-binding region in MazE, a series of N- and C-terminal deletions of the mazE gene were constructed in pGAD-C1, and cotransformed with the pGBD-MazF plasmid into the PJ69–4A cells, as shown in Fig. 7. To localize the MazE-binding region in MazF proteins, a series of N- and C-terminal deletions of the mazF gene were constructed in pGBD-C1 and cotransformed with the pGAD-MazE plasmid into the PJ69–4A cells. Assays of the interactions were performed by monitoring growth of cotransformants on synthetic dropout minimal medium (Clontech) lacking Trp, Leu, His, and adenine (Ade). The medium was supplemented with 1 mM 3-amino-1,2,4-triazole and incubated at 30 °C for 5 days.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
2 as determined by densitometer.
|
When (His)6MazE and MazF were mixed together and the mixture was
subjected to native PAGE, a new band appeared at position a near the top of
the gel (arrowhead a in Fig.
2). The gel corresponding to the new band was cut out and
incubated in a reducing buffer (20 mM Tris-HCl, pH 7.5, 100
mM NaCl, and 50 mM -mercaptoethanol) for 30 min at
room temperature, and then the gel was placed on the top of SDS-PAGE gel to
run a second dimensional electrophoresis to analyze the protein components.
After staining the gel with Coomassie Brilliant Blue, two bands corresponding
to (His)6MazE and MazF were observed, whereas (His)6MazE
moved slower than MazF on the SDS-PAGE. These results demonstrated that the
new band was the complex consisting of (His)6MazE and MazF. If the
gel cut from the native PAGE was not treated in the reducing buffer, three
protein bands were observed after it was subjected to the SDS-PAGE,
(His)6MazE, MazF, and the MazF dimer (data not shown). Three bands
appeared for the purified MazF on the native PAGE, but only one peak was
observed when the purified MazF protein was assayed by HPLC (data not shown).
The reason for the multi-band formation of MazF is not known at present.
|
Next, we attempted to determine whether the ratio of (His)6MazE
to MazF is stable in the complex. As shown in
Fig. 2A, different
amounts of (His)6MazE were added into the solution containing a
constant concentration of MazF (2 µM) to make the
(His)6MazE:MazF ratios as 1:1, 2:1, 4:1, 6:1, and 8:1, whereas in
Fig. 2B, different
amounts of MazF were added into the solution containing a constant
concentration of (His)6MazE (2 µM) to make the
(His)6MazE:MazF ratios as 1:1, 1:2, 1:4, 1:6, and 1:8. The mixtures
were incubated for 30 min at 4 °C and then analyzed by native PAGE. The
gel corresponding to the new band (at position a) was cut out and incubated in
the reducing buffer for 30 min at room temperature and then subjected to 15%
SDS-PAGE. The second dimensional gel was stained with Coomassie Brilliant Blue
to detect protein bands. Relative protein amounts in each lane were determined
by densitometer using purified (His)6MazE and MazF as controls. The
ratios of MazF to (His)6MazE in the complex were maintained almost
constant at 1.8 whenever (His)6MazE or MazF was added in excess in
the mixtures (Fig. 2). As
mentioned above, the MazE-MazF(His)6 complex was separated to MazE
and MazF(His)6 on a Tricine SDS-PAGE, and the ratio of
MazF(His)6 to MazE is 2
(Fig. 1, lane 2). The
molecular mass of the purified MazE-MazF(His)6 complex and MazF
were determined as 76.9 and 27.1 kDa by gel filtration with a SuperduxTM
200 column (Amersham Biosciences) (Fig.
3). MazF-(His)6 was purified from the
MazE-MazF(His)6 complex. MazF(His)6 was able to inhibit
the protein synthesis in an E. coli cell-free system (E.
coli T7 S30 extract system; Promega), and the protein synthesis was
rescued by the coaddition of (His)6MazE (data not shown). The
molecular mass of MazF-(His)6 was determined to be 28.3 kDa with
light scattering, suggesting that MazF(His)6 exists as dimer. The
structure of MazE has been demonstrated as a dimer
(30). Therefore, the
MazE-MazF(His)6 complex (76.9 kDa) may consist of one MazE dimer
(predicted to be approximately 18.6 kDa because the MazE molecular mass is 9.3
kDa) and two MazF(His)6 dimers (predicted to be 56.6 kDa).
|
MazF Enhances MazE Binding to the mazEF Promoter—The 50-bp
mazEF promoter fragment prepared as described under
"Experimental Procedures" was end-labeled by T4 polynucleotide
kinase with [-32P]ATP. Using EMSA, (His)6MazE,
MazF, and the MazE-MazF(His)6 complex were tested separately for
their binding abilities to the mazEF promoter DNA fragment.
(His)6MazE was able to shift the mazEF promoter fragment
at 2 µM or higher concentrations
(Fig. 4A, lanes
7–12). At 0.4–1.0 µM (His)6MazE, no
discrete mobility-shifted bands were observed, although the signals of the DNA
fragment started to smear upward (Fig.
4A, lanes 3–6), indicating that some
unstable (His)6MazE-DNA complexes were formed at these
concentrations. At 2–20 µM (His)6MazE, we
observed discrete mobility-shifted complexes, which move slower at the higher
concentrations of (His)6MazE
(Fig. 4A, lanes
7–12), suggesting that the number of (His)6MazE
molecules bound to the DNA fragment increased at higher (His)6MazE
concentrations. It is possible that there is more than one
(His)6MazE-binding site in the 50-bp mazEF promoter
fragment. On the other hand, MazF protein could not bind to the 50-bp
mazEF promoter DNA even at 20 µM concentration
(Fig. 4B). Increasing
amounts of both (His)6MazE and MazF proteins were added with a
constant (His)6MazE/MazF (1:2) ratio. Compared with
(His)6MazE alone, MazF significantly enhanced (His)6MazE
binding to the mazEF promoter. Under these conditions, the 50-bp
mazEF promoter fragment was shifted at a (His)6MazE
concentration of as low as 0.2 µM
(Fig. 4C), and
supershifting was observed at higher concentrations of the
(His)6MazE-MazF complex, which indicates that more
(His)6MazE-MazF complexes bind to the DNA fragment at higher
concentrations, suggesting there are multiple binding sites for the
(His)6MazE-MazF complex in the mazEF promoter.
|
Conserved Amino Acid Sequence in MazE Homologs—The MazE homologs were identified by the BLAST search, and their amino acid sequence alignments are shown in Fig. 5. Although generally MazE is not highly conserved in bacteria, there are still some conserved boxes in MazE. First, the N-terminal region of MazE is more conserved than other regions in MazE. MazE is an acidic protein with a pI of 4.7, but there are a few conserved basic residues (Lys7, Arg8, and Arg16) in its N-terminal region, named the N box (Fig. 5). Because MazE is able to bind the mazEF promoter DNA, the N box may be responsible for DNA binding. Secondly, there is a conserved C-terminal region, named the Hp box (Fig. 5), which contains several conserved hydrophobic residues.
|
The N Box of MazE Is Responsible for the DNA Binding of Both MazE and the MazE-MazF Complex—Various site-directed mutations were constructed in the mazE gene on pET21cc-EF(His)6 plasmid, converting the conserved amino acid residues in the N box to Ala. The complexes formed by MazE mutant proteins and MazF(His)6 were purified. These complexes were tested for their binding ability to the mazEF promoter DNA by EMSA. As shown in Fig. 6A, the complexes formed by MazE mutants with mutation in the N box (K7A, R8A, S12A, and R16A) and MazF(His)6 were unable to bind to the mazEF promoter DNA (Fig. 6A, lanes 3–6). However, the substitution mutations on the conserved amino acids outside the N box, such as MazE I43N and E57Q, did not affect the DNA binding of the complex (Fig. 6A, lanes 7 and 8, respectively). Various substitution mutations were also constructed in the mazE gene on pET28a-(His)6E plasmid. All of the (His)6MazE mutants with the substitution mutation in the N box (K7A, R8A, S12A, and R16A) lost their DNA binding ability (Fig. 6B, lanes 3, 4, 5, and 6, respectively), whereas the wild-type (His)6MazE was able to bind to the mazEF promoter (Fig. 6B, lane 2). The (His)6MazE mutants with the substitution mutation outside the N box (R48A, F53A, L55A/L58A, and E57Q) were able to bind the mazEF promoter DNA (data not shown). These results indicate that the DNA binding ability of the MazE-MazF complex is due to MazE protein in the complex and that the N box is responsible for the DNA binding of MazE.
|
Interaction between MazE and MazF—Yeast two-hybrid assays
were performed to examine the interaction between MazE and MazF. To
demonstrate which region of MazE is required for its interaction with MazF,
the full-length mazE gene and various N-terminal and C-terminal
deletion constructs of the mazE gene, as indicated in
Fig. 7, were generated by PCR
and cloned into the EcoRI-PstI sites of pGAD-C1 vector to
create in-frame translation fusions with the Gal4 transcriptional activation
domain, and then each of these plasmids was cotransformed with the pGBD-MazF
plasmid into PJ69–4A yeast cells, respectively. The cotransformants
harboring pGAD-MazE, pGAD-MazE(1–13),
pGAD-MazE(1–24), pGAD-MazE(1–37), or
pGAD-MazE(76–82) with pGBD-MazF were able to grow on the on
synthetic dropout medium (Clontech) lacking Trp, Leu, His, and Ade, whereas
the cotransformants harboring pGAD-MazE(1–46) or
pGAD-MazE(68–82) with pGBD-MazF were not. These data demonstrate
that the full-length MazE, MazE(1–13), MazE (1–24),
MazE(1–37), and MazE(76–82) are able to interact
with MazF, whereas the further N-terminal deletion mutant
MazE(1–46) and the further C-terminal deletion mutant
MazE(68–82) are unable to do so. These results indicate that the
region from residues 38 to 75 of MazE is responsible for the interaction with
MazF.
A series of truncation mutations from the N- and C-terminal ends of MazF were constructed in pGBD-C1 and cotransformed with pGAD-MazE into PJ69–4A cells. All of these cotransformed yeast cells were unable to grow on complete synthetic medium in the absence of Trp, Leu, His, and Ade, indicating that all of these MazF mutants were unable to interact with MazE. Therefore both N- and C-terminal regions of MazF may be involved in the interaction with MazE or the deletion mutations disrupt the MazF structure responsible for interaction with MazE. However, it is possible that the negative results of the yeast two-hybrid assays may be due to degradation of the fusion proteins.
Site-directed mutations were also performed on plasmid pET28a-(His)6E to construct (His)6MazE R48A, F53A, L55A/L58A, and E57Q mutants. The complex formations with these (His)6MazE mutants and MazF were examined by native PAGE. As shown in Fig. 8A, (His)6MazE mutants R48A, F53A, and E57Q were able to form the complex with MazF (Fig. 8A, lanes 5, 6, and 7, respectively), whereas the (His)6MazE L55A/L58A mutant was not (Fig. 8A, lane 4). By EMSA, it was found that both the wild-type (His)6MazE and the (His)6MazE L55A/L58A mutant were able to bind to the mazEF promoter DNA (Fig. 8B, lanes 2 and 3, respectively). When MazF was added, the wild-type (His)6MazE was able to interact with MazF to form the complex, resulting in a supershifted band near the top of the gel (Fig. 8B, lane 4) compared with the lane with wild-type (His)6MazE alone (Fig. 8B, lane 2). However, the addition of MazF to (His)6MazE L55A/L58A did not cause the supershifting of the DNA fragment, confirming that the (His)6MazE L55A/L58A mutant cannot interact with MazF to form a complex.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
(His)6MazE is able to interact with MazF and bind to the mazEF promoter. MazF(His)6, like MazF, forms a dimer and inhibits the in vitro protein synthesis, and the protein synthesis is rescued by coaddition of (His)6MazE (data not shown). Therefore the His tags appear to have no effects on the function of MazE and MazF in vitro. Using highly purified (His)6MazE and MazF, we demonstrated that (His)6MazE can bind to the mazEF promoter by itself and that the addition of MazF enhances (His)6MazE binding to the mazEF promoter DNA by more than 10-fold. At the higher concentrations of (His)6MazE or the (His)6MazE-MazF complex, supershifting is observed in the electrophoretic mobility shift assays with the mazEF promoter DNA, indicating that both (His)6MazE and the (His)6MazE-MazF complex have more than one binding site on the mazEF promoter DNA. A previous fluorescent study suggests that there may be three MazE-binding sides in the mazEF promoter region (30). It is interesting to note that the bands are not shifted in a stepwise manner. The reason for such shifting patterns is unknown at present. The site-directed mutations in the conserved N box of MazE (K7A, R8A, S12A, and R16A) disrupted the DNA binding ability of both (His)6MazE and the MazE-MazF(His)6 complex (Fig. 6), suggesting that MazE is responsible for the DNA binding ability of the MazE-MazF(His)6 complex and that the highly conserved N-terminal region in MazE is the DNA-binding domain. So far, the precise targets in the promoter sequence for the binding of MazE and the MazE-MazF complex have not been identified.
Yeast two-hybrid assays were performed to identify the region responsible for the MazE-binding to MazF. It was found that the region from residues 38 to 75 in MazE was required for its binding to MazF. There is a conserved C-terminal region in MazE named the Hp box, which is rich in hydrophobic residues. The Hp box mutations at the conserved Leu55 and Leu58 (L55A/L58A) disrupted the interaction between (His)6MazE and MazF. The yeast two-hybrid experiments also indicated that the entire structure of MazF protein may be required for its interaction with MazE, because deletions from either the N- or C-terminal end of MazF disrupted the interaction between MazE and MazF.
The molecular mass of the MazE-MazF(His)6 complex was determined
to be 76.9 kDa by gel filtration. When the purified MazE-MazF(His)6
complex was subjected to Tricine SDS-PAGE, the ratio of MazE to
MazF(His)6 was found to be 1:2
(Fig. 1, lane 2). Even
in the presence of excess amounts of (His)6MazE or MazF, the ratio
of (His)6MazE to MazF in the (His)6MazE-MazF complex was
stably maintained at approximately 1:1.8
(Fig. 2). Because both MazE
(30) and MazF(His)6
exist as dimers, the MazE-MazF(His)6 complex (76.9 kDa) may consist
of one MazE dimer (predicted to be approximately 18.6 kDa because the
molecular mass of MazE is 9.3 kDa) and two MazF(His)6 dimers
(predicted to be approximately 56.6 kDa because the molecular mass of
MazF(His)6 dimer is 28.3 kDa).
While preparing this manuscript, the crystal structure of the MazE-MazF complex was determined by Kamada et al. (31). The crystal structure of the MazE-MazF complex confirmed our results in its following aspects: 1) In the crystal structure, MazE and MazF form a 2:4 heterohexamer, consisting of alternating MazF and MazE homodimers (MazF2-MazE2-MazF2). It is important to note that the 2:4 stoichiometric complex formation between MazE and MazF appears to be very stable, because the ratio between (His)6MazE and MazF in the (His)6MazE-MazF complex was found irrespective of which protein was added in large excess (Fig. 2). 2) The C-terminal region of MazE interacts with the MazF homodimer in the structure of MazE-MazF complex. The Hp box region identified in this study is involved in the seemingly most stable interface between MazE and MazF (Fig. 9). The side chains of hydrophobic amino acid residues (Leu55, Leu58, Val59, and Ile62) in the Hp box contact a cluster of hydrophobic residues in the MazF homodimer. Indeed (His)6MazE L55A/L58A mutant was not able to form a complex with MazF (Fig. 8), suggesting that these hydrophobic interactions are essential for the MazE-MazF complex formation. 3) Based on the similarity between MazE and other addiction module antidotes and the distribution of the basic regions on the electrostatic surfaces of MazE and MazF, Kamada et al. (31) proposed that Lys7 and Arg8 in MazE serve as the primary DNA anchoring sites in the MazE-MazF complex. In the present paper, we showed that the DNA binding abilities of (His)6MazE and the MazE-MazF(His)6 complex were disrupted not only by the site-directed mutations at Lys7 and Arg8 but also by mutations at other conserved amino acid residues (Ser12 and Arg16) in the N box (Fig. 9). It is possible that, because MazE exists as a dimer, the two N boxes in the MazE dimer may be involved together in DNA binding.
|
The coexpression of MazE and MazF is not only negatively autoregulated by MazE and MazE-MazF complex but also inhibited by ppGpp. It has been demonstrated that the global regulatory nucleotide ppGpp affects the activity of RNA polymerase on specific promoters. A conserved GC-rich consensus sequence localized immediately downstream of the –10 promoter element has been recognized as an important cis-element for negative stringent control (32). A GC-rich sequence (GCGG) exists immediately downstream of the –10 box of the major mazEF promoter P2 (24). It is possible that the inhibition of mazEF expression by ppGpp is due to its effect on RNA polymerase, whereas the autoregulation of the mazEF operon by MazE and the MazE-MazF complex may be due to their binding to the mazEF promoter region to block RNA polymerase from binding to the promoter. At present, there is no evidence suggesting that ppGpp has any effects on the protein-protein or protein-DNA interaction within the mazEF addiction module.
The cellular effects of the toxins in the addiction modules have been studied quite extensively. CcdB, the toxin in the ccdA-ccdB system, interacts with DNA gyrase to induce DNA cleavage and block DNA replication (9, 13), and RelE, the toxin in the relBE system, has a ribosome-dependent codon-specific mRNA cleavage activity at the ribosome A site (21). MazF has been shown to block translation as well as DNA replication (20). The detail mechanism of MazF function is currently under investigation.
|
FOOTNOTES |
|---|
To whom correspondence should be addressed: Dept. of Biochemistry, Robert Wood
Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Tel.:
732-235-4115; E-mail:
inouye{at}rwja.umdnj.edu.
1 The abbreviations used are: ppGpp, guanosine
3',5'-bispyrophosphate; EMSA, electrophoretic mobility shift
assay; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  K. C. Rice and K. W. Bayles Molecular Control of Bacterial Death and Lysis Microbiol. Mol. Biol. Rev., March 1, 2008; 72(1): 85 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Fu, N. P. Donegan, G. Memmi, and A. L. Cheung Characterization of MazFSa, an Endoribonuclease from Staphylococcus aureus J. Bacteriol., December 15, 2007; 189(24): 8871 - 8879. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Daines, M. H. Wu, and S. Y. Yuan VapC-1 of Nontypeable Haemophilus influenzae Is a Ribonuclease J. Bacteriol., July 15, 2007; 189(14): 5041 - 5048. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Moritz and P. J. Hergenrother Toxin-antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci PNAS, January 2, 2007; 104(1): 311 - 316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mochizuki, K. Yahara, I. Kobayashi, and Y. Iwasa Genetic Addiction: Selfish Gene's Strategy for Symbiosis in the Genome Genetics, February 1, 2006; 172(2): 1309 - 1323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Senn, P. Giachino, D. Homerova, A. Steinhuber, J. Strassner, J. Kormanec, U. Fluckiger, B. Berger-Bachi, and M. Bischoff Molecular Analysis and Organization of the {sigma}B Operon in Staphylococcus aureus J. Bacteriol., December 1, 2005; 187(23): 8006 - 8019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Cherny, L. Rockah, and E. Gazit The YoeB Toxin Is a Folded Protein That Forms a Physical Complex with the Unfolded YefM Antitoxin: IMPLICATIONS FOR A STRUCTURAL-BASED DIFFERENTIAL STABILITY OF TOXIN-ANTITOXIN SYSTEMS J. Biol. Chem., August 26, 2005; 280(34): 30063 - 30072. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Lah, M. Simic, G. Vesnaver, I. Marianovsky, G. Glaser, H. Engelberg-Kulka, and R. Loris Energetics of Structural Transitions of the Addiction Antitoxin MazE: IS A PROGRAMMED BACTERIAL CELL DEATH DEPENDENT ON THE INTRINSICALLY FLEXIBLE NATURE OF THE ANTITOXINS? J. Biol. Chem., April 29, 2005; 280(17): 17397 - 17407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhao and R. D. Magnuson Percolation of the Phd Repressor-Operator Interface J. Bacteriol., March 15, 2005; 187(6): 1901 - 1912. [Abstract] [Full Text] [PDF]
|
|
