1 Unité de Biochimie Microbienne, Institut Pasteur, URA 2172 du Centre National de la Recherche Scientifique, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
2 Laboratoire de Microscopie Cellulaire et Moléculaire, Institut Gustave Roussy, UMR 1598 du Centre National de la Recherche Scientifique, 94805 Villejuif Cedex, France
3 Groupe de Microscopie Structurale Moléculaire, Institut Pasteur, URA 2185 du Centre National de la Recherche Scientifique, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
4 Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, Boston, MA 02111, USA
5 Laboratoire de Minéralogie Cristallographie, Université Paris 6, UMR 7590, IPGP, CNRS Case 115, Tour 16, 4 Place Jussieu, 75252 Paris Cedex 05, France
Correspondence
Michel Débarbouillé
mdebarbo{at}pasteur.fr
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ABSTRACT |
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INTRODUCTION |
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In B. subtilis, arginine is converted to glutamate by the three-step arginase pathway. The relevant enzymes are encoded by the rocABC and rocDEF operons (Calogero et al., 1994; Gardan et al., 1995
). Arginine is cleaved by arginase (RocF) to give ornithine, which is then converted to glutamate semialdehyde by ornithine aminotransferase (RocD). Finally, the conversion of glutamate semialdehyde to glutamate is catalysed by a pyrroline-5-carboxylate dehydrogenase (RocA). This last reaction is shared with the proline catabolic pathway. RocG (glutamate dehydrogenase) catalyses the conversion of glutamate to 2-oxoglutarate, the final step in the utilization of arginine, ornithine and proline as carbon or nitrogen sources (Belitsky & Sonenshein, 1998
). The RocR protein positively regulates the rocABC and rocDEF operons and the rocG gene (Belitsky & Sonenshein, 1999
; Calogero et al., 1994
; Gardan et al., 1995
, 1997
).
Expression of rocG, rocABC and rocDEF is 54-dependent (Belitsky & Sonenshein, 1998
; Calogero et al., 1994
; Gardan et al., 1995
). Expression of these genes is fully induced by arginine or ornithine in the growth medium. An intermediate level of expression was observed with proline (Belitsky & Sonenshein, 1998
; Gardan et al., 1997
). RocR is a member of the NtrC/NifA family of proteins that bind to enhancer-like elements often referred to as upstream activating sequences (UASs). Members of this family of activators contain a region of 220240 residues called the central domain, which is specifically required for the formation of open complexes between
54-containing RNA polymerase and
54-dependent promoters, also known as -12, -24 promoters (Kustu et al., 1989
, 1991
; Merrick, 1993
; Morett & Segovia, 1993
).
A DNA region essential for rocABC transcription was identified by deletion mapping near position -165 with respect to the transcription start site. This region includes a palindromic sequence of 17 bp that may serve as a UAS. A second copy of this sequence is present near position -122 of the promoter region. We designated these two sequences UAS1 and UAS2, respectively, and suggested that RocR acts by binding to these 17 bp UASs to stimulate transcription of rocABC (Calogero et al., 1994). Similar sequences appear twice within the rocDEF upstream region as well (Gardan et al., 1995
). The rocG gene, located just upstream from rocABC, is transcribed by
54-containing RNA polymerase and also requires RocR for its transcription. The rocG gene has no UAS, however. Its expression depends instead on a sequence designated downstream activating sequence (DAS) located 1·5 kb downstream of the rocG -12, -24 promoter. The same DNA region includes the rocABC UAS1 and UAS2 (Belitsky & Sonenshein, 1999
).
In the present work, RocR was purified and shown to bind specifically to the intergenic region between rocG and the rocABC operon. DNaseI footprinting experiments showed that RocR binds to two sites that correspond closely to UAS1 and UAS2. Point mutations in the palindromic sequences affected transcription of both rocG and rocABC. To function, these UAS/DAS sequences must be brought into close proximity with the -12, -24 promoters upstream from rocG and rocABC, suggesting that DNA conformation plays a role during the activation process. Electron microscopy analysis indicated that this region contains an intrinsic curvature centred between the UAS/DAS enhancer element and the rocABC promoter.
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METHODS |
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DNA manipulation.
Standard procedures were used to extract plasmids from E. coli (Sambrook et al., 1989). Restriction enzymes, phage T4 DNA polymerase, phage T4 DNA ligase and T4 polynucleotide kinase were used as recommended by the manufacturers. DNA fragments were purified from the agarose gel with a Prep-A-Gene kit (Bio-Rad).
Plasmids constructions.
Plasmid pNO11 was constructed as follows. A 1·7 kbp DNA fragment containing rocG and the beginning of rocA was amplified by PCR using the Pfu polymerase and two oligonucleotides, OMD41b and OMD42b. Oligonucleotides used for PCR included mismatches allowing the creation of EcoRI and BamHI restriction sites. The mutagene M13 in vitro mutagenesis kit (Bio-Rad) was used, based on the procedure described by Kunkel et al. (1987). The 1 kb EcoRIBamHI DNA fragment was cloned between the EcoRI and BamHI restriction sites of the replicative form of M13mp19 to give phage M13mp19 rocG rocA. Mutations were created as described previously (Martin-Verstraete et al., 1992
) using oligonucleotides OMD49 (5'-GAG AAG CCT CCA CAA AAT AAT TTT GC-3'), OMD50 (5'-GCC TCC GCA AAA TAA TTT TAC ATT TG-3'), OMD59 (5'-GCC TCC ACA AAA TAA TTT TAC ATT TG-3'), OMD60 (5'-GCC TCC GAA AAA TAA TTT TGC ATT TG-3'), OMD61 (5'-GCC TCC GCA AAA TAA TTT TGA ATT TG-3'), OMD62 (5'-GCC TCC GCA AGG TAA TTT TGC ATT TG-3') and OMD63 (5'-GCA AAA AAC ACA AAAT AAA TTT ACG TTC AAG-3'), containing one or several mismatches. The presence of the mutations was verified by DNA sequencing and the EcoRIBamHI DNA fragment was then cloned between the corresponding sites of vector pDIA5307 just upstream from a promoterless lacZ gene.
Plasmid pNO12 was used to introduce a deletion replacement of the UAS/DAS region in B. subtilis 168. This plasmid was constructed by cloning a 500 bp EcoRIBamHI DNA fragment, a 1 kb BamHIPstI DNA fragment carrying the kanamycin-resistance gene and a 500 bp PstIHindIII DNA fragment between the EcoRI and HindIII sites of pHT181 (Lereclus & Arantès, 1992), to give pNO12. The 500 bp EcoRIBamHI DNA fragment and the 500 bp PstIHindIII DNA fragment correspond, respectively, to the chromosomal DNA region encoding the end of rocG and the beginning of rocA and were generated by PCR using oligonucleotides OMD51, OMD52 and OMD53, and OMD54, respectively. Plasmid pNO12 was used to transform B. subtilis 168. Kanamycin-resistant, erythromycin-sensitive integrants arose through a double crossover event in which the entire UAS/DAS region was deleted and replaced with the kanamycin-resistance gene. The corresponding mutant was designated QB7705.
The previously constructed plasmid pDIA5326 (Calogero et al., 1994) contains the promoterless lacZ gene fused to the EcoRISalI fragment of the rocGrocA region containing 0·75 kb of the rocG sequence, the UAS, the rocA promoter and 0·3 kb of the rocA sequence. Plasmid pBB1085 was constructed by deleting the 0·74 kb SmaISacII fragment of pDIA5326 and contains the UAS and the rocA promoter but no rocG sequence. Plasmid pBB1087 was constructed by deleting the 0·85 kb SmaIBclI fragment of pDIA5326 and contains the rocA promoter but no UAS or rocG sequence. Plasmid pET28a was used for protein overexpression and purification (Novagen).
RocR was overproduced using pETRocR. This plasmid was constructed by cloning a 1391 bp NcoIXhoI DNA fragment corresponding to the rocR coding sequence between the NcoI and XhoI sites of pET28a. This DNA fragment was generated by PCR using oligonucleotides TM-268 (5'-CCACCATGGTCAAAGACAGCGAATTCCTCACATTG-3') and TM-269 (5'-CTCCTCGAGTTCGTTAGACGAGTGAGAAAAT-3'), thus replacing the original GTG translational initiation codon with an ATG codon, and the TAA stop codon by the XhoI restriction site. This allows the creation of a translational fusion adding six carboxy-terminal histidine residues to the RocR protein and placing expression of the gene under the control of a T7 bacteriophage promoter.
Overproduction and purification of RocR.
Plasmid pETRocR was used to transform E. coli BL21DE3, in which the T7 RNA polymerase gene is under the control of the inducible lacUV5 promoter (Studier & Moffatt, 1986
). The resulting strain was grown in 1 l LB medium at room temperature until the mid-exponential phase (OD600 value of approx. 0·8), IPTG was added (1 mM) and incubation pursued for 3 h. The cells were centrifuged at 10 810 g for 30 min, and resuspended in 1/50 of the culture volume of buffer I [50 mM NaH2PO4 (pH 8), 300 mM NaCl, 20 mM imidazole]. The cells were disrupted by sonication and cell debris was removed by two consecutive centrifugation steps at 17 210 g for 30 min. E. coli crude protein extracts were loaded onto a 0·15 ml Ni-NTA agarose (Qiagen) column previously equilibrated with buffer I. The column was washed with 10 volumes of buffer II [50 mM NaH2PO4 (pH 6), 300 mM NaCl, 30 mM imidazole] and RocR was eluted with an imidazole gradient (30500 mM). Fractions were pooled and dialysed against buffer III [50 mM NaH2PO4 (pH 8), 300 mM NaCl, 50 % (v/v) glycerol] to remove the imidazole and concentrate the protein solution approximately fourfold.
SDS-PAGE on 12 % acrylamide gels was performed as described previously; gels were stained with Coomassie blue (Laemmli, 1970). Protein concentrations were determined using the Bio-Rad protein assay (Bradford, 1976
).
Gel mobility shift and DNA-binding assay.
A 263 bp EcoRIBamHI DNA fragment, corresponding to the promoter region of the rocABC operon (positions -267 to -25 with respect to the transcription initiation site), was generated by PCR using oligonucleotides RA1 (5'-GAAGAATTCGTGGATATGCGTTTGGCGGC-3') and RA2 (5'-CGCGGATCCGCCATTTTCTTGTCTTTTGC-3'). This DNA fragment was radioactively labelled by BamHI digestion and treatment with the Klenow fragment of DNA polymerase I (Roche) in the presence of a mixture of dGTP, dCTP, dTTP (0·08 mM) and [-32P]dATP (40 µCi; 148 kBq). Binding of RocR to DNA was carried out in a 10 µl reaction mixture containing 104 c.p.m. [
-32P]-labelled DNA, 1 µg poly-(dI-dC)
poly-(dI-dC) (Amersham-Pharmacia), 25 mM NaH2PO4 (pH 7), 150 mM NaCl, 0·1 mM EDTA, 2 mM MgSO4, 1 mM DTT and 10 % (v/v) glycerol. The DNA-binding reaction was initiated by the addition of RocR and was incubated at room temperature for 20 min. Samples were then directly loaded onto a 4 % polyacrylamide gel (89 mM Tris/borate, 2 mM EDTA) during electrophoresis (14 V cm-1). Electrophoresis was pursued for 1 h at room temperature and the gels were then dried and analysed by autoradiography.
DNaseI footprinting.
DNA fragments used for DNaseI footprinting were prepared by PCR, using the Pfu polymerase (Stratagene) and 20 pmol primers RA1 and RA2, one of which was previously labelled with T4 polynucleotide kinase (New England Biolabs) and [-32P]dATP. Labelled PCR products were purified using the Qiaquick PCR purification kit (Qiagen). RocR binding to DNA was carried out as described above, with the addition of BSA (0·1 µg), and DNaseI footprinting experiments were then performed as described previously (Derré et al., 1999
).
-Galactosidase and glutamate dehydrogenase assays.
B. subtilis cells containing lacZ fusions were grown to an OD600 value of 1. -Galactosidase specific activities were determined as described previously and are expressed as Miller units per milligram of protein or as Miller units per OD600 (Miller, 1972
).
Glutamate dehydrogenase was assayed, as described previously, in strains carrying a gudB : : tet mutation, to avoid any contribution from the GudB glutamate dehydrogenase (Belitsky et al., 1998). The values reported represent a mean of at least two independent assays.
DNA-fragment preparation for electron microscopy.
All DNA fragments used in electron microscopy experiments were obtained by PCR using the Pwo polymerase and the following oligonucleotides: OMD145 (5'-GGAAGAACACTGAAAGAAACACAGATTTTAAACG-3'); OMD141 (5'-CCTAGCGAGTGCCGGCGGCGTCACGGTTTC-3'); OMD140 (5'-GCATCGCTTTTTCAGCAAGCTCTTGATCCGCTGTAG-3').
OMD140 was biotinylated at the 5' end. The oligonucleotides were purified by an anion exchange MonoQ column using a SMART system (Pharmacia). The 696 bp biotinylated fragment (containing the two UAS sites and rocABC promoter amplified using OMD140 and OMD141) was dimerized using streptavidin. This technique allowed us to obtain specifically oriented tail-to-tail DNA dimers. A similar strategy was used to synthesize the 2131 bp DNA fragment using oligonucleotides OMD140 and OMD145. This DNA fragment contains the promoter of rocG, rocG and the promoter of rocABC.
RocRDNA complex formation.
Purified RocR was incubated for 10 min at 20 °C at different ratios with the 2131 bp DNA fragment in binding buffer containing 10 mM Tris/HCl (pH 7·5), 50 mM NaCl and 5 mM MgCl2. DNA was maintained at 2 nM and complexes were formed at 10, 20, 40 and 100 nM of RocR. Complexes were purified by Superose 6B chromatography (APbiotech).
Observation by electron microscopy.
After purification, 5 ml of a solution containing 0·5 mg DNA ml-1 were deposited onto a 600 mesh copper grid covered with a very thin carbon film activated by a glow discharge in the presence of pentylamine (Dubochet et al., 1971). Grids were washed with 2 % aqueous uranyl acetate, dried and observed in annular dark-field mode, using a Zeiss 902 electron microscope (Le Cam & Delain, 1995
).
Acquisition and digitization of the molecules.
For each orientation, 200 images of individual dimer molecules showing unambiguous trajectories were taken and stored on a hard disk through the use of a camera coupled to the electron microscope. Pictures at a magnification of 140 000x times; were digitized on an image analyser (IBAS Kontron) by following the contour of the molecule and picking up 420440 points.
Curvature analysis.
Experimental curvature maps of DNA fragment were obtained from the analysis of the set of 200 molecules. The co-ordinates (x, y) of each molecular pathway were smoothed and iso-segmented according to the real base-pair number of the DNA. Using the DNA ResCue program (Larquet et al., 1995), the curvature was quantified by the SD parameter, ratio of the curvilinear (S) to end-to-end (D) lengths of the fragment within a 60 bp window moving along the DNA by 10 bp (Muzard et al., 1990
). This ratio gives the w parameter, mean dinucleotide wedge angle per base pair, which is an intrinsic parameter for DNA curvature, independent of the fragment length.
DNA trajectory modelling.
The spatial sequence path of the DNA helix axis was modelled with the DNA ResCue program. A local co-ordinate system is associated to each base pair. Its origin is on the helical axis. The x and y axes are, respectively, the transversal and longitudinal axis in the base-pair plane, and the z axis points to the next base pair. The local co-ordinate system linked to the (n+1)th base pair is deduced from the (n)th base-pair co-ordinate system by applying a unit z-translation, and three successive rotations: twist around the z axis, roll around the y axis so formed, and tilt around the x axis resulting from the two previous rotations. Twist angles (Kabsch et al., 1982) and roll and tilt angles were taken from the curvature model considered. Calculations were performed using transformation matrices that refer to the centre of the co-ordinate system of each base pair in the co-ordinate system associated with the first base pair. The three-dimensional co-ordinates of the centres of the successive base pairs determine the path of the DNA helix axis.
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RESULTS |
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A comparison by SDS-PAGE of crude extracts from E. coli cells carrying the vector pET28a alone or the plasmid pETRocR showed an overproduced RocR-specific band of the expected molecular mass (approx. 53 kDa) (Fig. 1, lanes 2 and 3). His-tagged RocR protein was purified in a single step using a Ni-NTA agarose column (see Methods) and SDS-PAGE analysis revealed a high degree of purity (Fig. 1
, lane 4).
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Mutations affecting rocABC and rocG expression
To define precisely the DNA sequence important for the activation of rocABC and rocG, point mutations were introduced into the dyad symmetry elements of UAS1 and UAS2. A DNA fragment containing the end of rocG, the entire rocA promoter region and the two UASs was cloned upstream from lacZ in plasmid pDIA5307. Point mutations were introduced by site-directed mutagenesis in each copy of the two UASs. The resulting plasmids were subsequently introduced by transformation into a B. subtilis strain containing a deletion of the two UASs and the rocABC promoter region. In the recombinant strains, the plasmids integrated by a single crossover event via homologous sequences at the end of the rocG gene. As a result, UAS1 and UAS2 were placed between the end of rocG and the promoter of the rocA'lacZ transcriptional fusion (Fig. 4a). The two 17 bp palindromic sequences are centred at positions -168 (UAS1) and -124 (UAS2) with respect to the rocABC transcription start site, and each inverted repeat contains an invariant GC dinucleotide. As shown in Table 2
, single mutations in the GC doublet of UAS1 caused a two- to fivefold decrease in
-galactosidase levels compared to the parental strain. Mutants were constructed in which two mutations were introduced in a polyadenine region in the middle of UAS1 (strain QB7712) or both GC doublets of UAS1 were modified (strain QB7709). Expression of rocA'lacZ in these two strains was also lowered two- to fivefold, respectively (Table 2
). Mutations modifying the conserved GC doublets of UAS2 (strain QB7715) also led to a significant decrease in lacZ expression (Table 2
), indicating that both UASs are required for full rocABC expression. To test if the two UASs act in transcriptional activation of rocABC, we constructed a mutant affected in all four GC doublets (strain QB7716). As shown in Table 2
, the combination of these four mutations completely abolished rocABC expression.
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Relocation of the UAS/DAS downstream from rocA
Unlike other 54-dependent promoters, rocG has no UAS and its expression depends on a sequence located 1·5 kb downstream from its promoter. This sequence still retains partial activity and can activate rocG if moved upstream from its promoter or 15 kb downstream (Belitsky & Sonenshein, 1999
). To determine whether this activation at a distance is specific to the rocG promoter, relocation experiments were carried out to move the UAS/DAS region downstream from the integrated pBB1085. Plasmid pBB1085 contains the two UASs, the rocA promoter and the beginning of rocA. Strain BB1662 contains a chromosomal copy of the rocA promoter lacking UAS1 and UAS2. When introduced into strain BB1662, pBB1085 can only integrate into the chromosome downstream of the UAS deletion. As a result, the two UASs of pBB1085 are placed downstream from the rocAlacZ fusion in strain BB1672 and are separated from the rocA promoter by the entire length of pBB1085 (9·1 kbp) (Fig. 4b
). Results of
-galactosidase assays are shown in Table 3
. In strain BB1672, rocA'lacZ expression levels represent 15 % of that of the wild-type strain (BB1648). This intermediate level is RocR-dependent, as shown in strain BB1673. As a control, plasmid pBB1087, identical to pBB1085 but lacking the two UAS/DAS, was integrated in the chromosome of strain BB1662, leading to strain BB1889, in which rocA'lacZ expression is virtually abolished. Taken together, these experiments indicate that the two UASs relocated downstream from the rocA gene are still able to allow RocR-dependent activation of transcription from the -12, -24 promoter.
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DISCUSSION |
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The two octanucleotide inverted repeat sequences are separated by a single base pair, thus placing them on the same face of the DNA double helix. The inverted repeat sequence is present twice within the rocGrocABC intergenic sequence, constituting two UAS/DAS, both of which are required for full expression of rocG and the rocABC operon. Binding to both UASs appears to occur simultaneously, as no intermediate DNAprotein complexes are detected by gel mobility shift experiments (Fig. 2) as RocR concentrations are varied, and in DNaseI footprinting protection is not progressive but is instead immediate and simultaneous for both UAS elements. This suggests that RocR has the same affinity for each site (Fig. 3
). This is in agreement with the mutational analysis, since single- or double-point mutations in either of the inverted repeats diminish rocABC and rocG expression, indicating that the UAS and the DAS are the same regulatory elements. Gel mobility shift and DNaseI footprinting experiments performed in the presence of 10 mM ornithine or arginine showed no difference in RocR binding, suggesting that the protein does not require the inducer to bind to DNA in vitro (data not shown). This confirms previous results indicating that RocR is able to repress its own expression in vivo in an inducer-independent fashion and suggests that the role of the inducer is to alter the conformation of RocR so that it can interact with
54 RNA polymerase (Gardan et al., 1995
).
We have previously reported that deletion of half of UAS1 led to a partial inhibition of rocABC transcription (Calogero et al., 1994). A strong inhibition was only observed in strains containing a deletion of both UASs. These results led to the assumption that RocR multimerizes on both UAS/DAS and that mutations in both sites are required to abolish transcription activation by RocR. A similar situation was described for the interaction of NtrC with its cognate UAS, with dimers of the protein binding co-operatively to the enhancer element, which consists of two specific binding sites. Co-operative binding of NtrC results from interaction between NtrC dimers. Moreover, it was also shown that transcription activation by NtrC at the glnA promoter might depend on formation of an oligomer larger than a tetramer in which additional dimers are associated through proteinprotein interactions with those directly bound to the enhancer (Wyman et al., 1997
). Multimerization to higher forms is a common feature of all NtrC-like proteins tested so far (Farez-Vidal et al., 1996
; Perez-Martin & De Lorenzo, 1996
; Wikstrom et al., 2001
; Wilson et al., 1994
; Wyman et al., 1997
).
54-Dependent promoters are activated at a distance by specific regulators bound to UASs that must loop out for productive contacts with the
54-RNA polymerase holoenzyme. The function of the enhancer is to tether the regulator at a high local concentration near the promoter. Studies of activation of
54-dependent transcription have shown that looping can be effected by random or transient changes in DNA conformation or can be directed by a site-specific DNA-binding protein such as integration host factor (IHF). In Gram-negative bacteria, an IHF site is often found between the binding site for the activator and the
54-RNA polymerase-binding site. However, no equivalent of IHF has been found to be encoded in the B. subtilis genome.
DNA fragments encompassing the rocA promoter region migrate anomalously on polyacrylamide gels and it was suggested that this effect is caused by an intrinsic bend in the DNA (Miller et al., 1997). Electron microscopy experiments indicate unambiguously that a strongly curved region is indeed observed upstream from the -12, -24 promoter of rocABC. Binding of RocR also appears to induce bending of the DNA, since several enhanced DNaseI cleavage sites appear within the protected regions constituting UAS1 and UAS2 (Fig. 3
). These results suggest a molecular basis for the contact between the RocR activator bound at the UAS/DAS elements and the RNA polymerase located at the rocABC promoter. However, the intrinsic curvature located between the UAS/DAS region and the -12, -24 promoter of rocABC does not explain the simultaneous activation at a distance of the rocG promoter. One possibility is that RocR binds with high affinity to strongly curved DNA as described for HNS (Thei Dame et al., 2001
). Oligomerization of RocR along the DNA starting from its initial nucleation-binding site might bridge the adjacent UAS/DAS segment. Such a structure may facilitate activation of both promoters by RocR. The role of an additional regulatory protein, AhrC, remains unclear. AhrC is similar to the E. coli ArgR repressor and is a positive regulator of the synthesis of the enzymes of the arginine catabolic pathway (Harwood & Baumberg, 1977
). It has been previously shown that AhrC makes contacts with the -12, -24 region of the rocABC promoter (Miller et al., 1997
). Since AhrC is also required for the activation of rocG expression (Belitsky & Sonenshein, 1999
), it could be involved in the stabilization of a putative DNA loop, including the rocG and the rocABC promoters.
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ACKNOWLEDGEMENTS |
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Received 23 September 2002;
revised 11 December 2002;
accepted 19 December 2002.
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