Department of Bacteriology, University of Wisconsin, 1710 University Avenue, Madison, WI 53726-4087, USA
Correspondence
Jorge C. Escalante-Semerena
escalante{at}bact.wisc.edu
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ABSTRACT |
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Present address: Department of Microbiology, University of Iowa, IA 52246, USA.
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INTRODUCTION |
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In Salmonella enterica serovar Typhimurium LT2 (hereafter referred to as S. enterica), functions required for catabolism of propionate via the 2-methylcitric acid cycle (Fig. 1b) are encoded by the prp locus (Horswill & Escalante-Semerena, 1997
). The prp locus comprises two divergently transcribed units. One unit, prpBCDE, encodes most of the enzymes needed to convert propionate to pyruvate, with aconitase activity being also required (Horswill & Escalante-Semerena, 2001
). A second transcriptional unit (prpR) encodes a
54-dependent transcriptional activator (Horswill & Escalante-Semerena, 1997
) (Fig. 1a
).
54-dependent transcriptional activators belong to the AAA+ superfamily of mechanochemical ATPases (Zhang et al., 2002
). These activators display a clear three-domain structure, with a highly variable N-terminal signal-sensing (A) domain, a catalytic (C) domain with ATPase activity, and a C-terminal DNA-binding (D) domain (Fig. 2
) (Morett & Segovia, 1993
). The linker region between the sensing (A) and catalytic (C) domains plays an active role in intramolecular signal transduction in certain members of this family of activators (Garmendia & de Lorenzo, 2000
; O'Neill et al., 2001
).
54-dependent activators typically bind to an upstream sequence known as the Enhancer-like Element (ELE) (Xu & Hoover, 2001
), and require a signal for activation. The signal can be the phosphorylation of the activator by a cognate sensor kinase, proteinprotein interactions, or the binding of a small-molecule co-activator.
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METHODS |
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Recombinant DNA procedures
DNA isolation and manipulations.
Cells were transformed by the CaCl2 heat-shock procedure or by electroporation, as described by Ausubel et al. (1989). Plasmid DNA was purified from cultures incubated at 37 °C for 1618 h using the Wizard SV DNA purification system (Promega). Restriction enzymes were purchased from Promega, MBI Fermentas or New England Biolabs. All enzymes were used according to manufacturers' instructions.
PCR.
All DNA amplifications were performed in a 2400 GeneAmp PCR system (Perkin-Elmer). EnzyPlus DNA polymerase (Enzypol) was used in all amplifications. Primers were obtained from IDT DNA Technologies.
DNA fragment and digested plasmid purifications.
DNA fragments and plasmids were purified after restriction digestion or PCR amplification by agarose gel purification. DNA was recovered from excised agarose bands using the Qiagen Gel Purification kit.
Ligation reactions.
All ligation reactions were performed using T4 DNA ligase (MBI Fermentas) following manufacturer's instructions.
DNA sequencing.
All constructed plasmids were sequenced using the ABI PRISM dye terminator system (Perkin-Elmer Life Sciences) in accordance with the manufacturer's instructions. Sequencing reactions were cleaned using CleanSeq magnetic beads (Agencourt). Sequencing was performed at the Biotechnology Center of the University of Wisconsin-Madison.
Plasmid constructions
Plasmid pPRP64.
This plasmid encodes a protein fused at its N-terminus to glutathione-S-transferase (GST) from Schistosoma japonicum. The prpR gene from plasmid pPRP8 (prpRBCDE+) was PCR-amplified using primers PrpR 5'-BamHI (5'-TAG GAT CCA TGA CGA CTG CCC ACA-3') and PrpR 3' (5'-CCT CTA GAA TTT GTC TTA ATT ATC-3') and inserted in the BamHI and SmaI of plasmid pGEX-2t (Amersham Pharmacia). Insertion of a BamHI site immediately after the initiation codon allowed for in-frame cloning of the prpR gene with the GST N-terminal fusion protein reading frame, yielding plasmid pPRP64.
Plasmid pPRP157.
The prpR gene was PCR-amplified from pPRP8 with primers PrpR5' RBS-NdeI (5'-CCT TGG ACA TAT GAC GAC TGC CCA CA-3') and PrpR3'SmaI-nostop (5'-ACG CCC GGG ATT ATC CGA CTG GTC TTT-3') and inserted in the NdeI and SmaI sites of plasmid pTYB2 (New England Biolabs). The resulting plasmid was referred to as pPRP157.
Plasmid pPRP158.
A 990 nt fragment of prpR encoding the 330 C-terminal amino acids of PrpR (prpR234) was amplified using the primers 5'NdeI-A (5'-CCT TGG ACA TAT GGG ATT ACA AAC CCG GTA T-3') and PrpR-3'SmaI-nostop (see above), and the amplified fragment was inserted in the NdeI and SmaI sites of pTYB2 to yield plasmid pPRP158.
Plasmid pPRP160-161.
The prpR alleles encoding PrpRA162T (prpR238) and PrpRR24S (prpR245) were PCR-amplified from the respective mutagenized pPRP56 clone (Palacios & Escalante-Semerena, 2000) using primers prpR5'RBS-NdeI (5'-CCT TGG ACA TAT GAC GAC TGC CCA CA-3') and PrpR-3'SmaI-ns (5'-ACG CCC GGG ATT ATC CGA CTG GTC TTT-3') and inserted in the NdeI/SmaI sites of plasmid pTYB2. Plasmid pPRP160 encoded the prpR allele PrpRA162T, whereas plasmid pPRP161 encoded allele PrpRR24S.
Plasmid pPRP164.
A 405 bp fragment containing half of prpR and the prpB promoter was amplified with primers pPRP60seqEcoRI (5'-TAA GAA TTC ATG GTC GCC AGA TGA TCG-3') and prpB prom-HindII (5'-CGC AAA GCT TCC TCA TGT TAG TAA ATT G-3') into the EcoRI and HindIII sites of p770 (Schneider et al., 2002). This fragment contains the prpB promoter 5' of a fragment of rrnB followed by two strong terminators.
Purification of PrpR protein.
The wild-type prpR allele in plasmid pPRP64 was overexpressed and PrpR protein was purified. This plasmid overexpresses a GSTPrpR protein fusion. A 5 ml culture was inoculated with a freshly transformed colony and grown overnight at 37 °C with shaking. This culture was used to inoculate 1·5 l LB containing 50 µg ampicillin ml1, which was grown with slow shaking at 37 °C until an OD650 of 0·5 was reached. The culture was transferred to a 15 °C bath, and prpR expression induced with 0·4 mM IPTG for 24 h. Cells were collected by centrifugation and resuspended in 15 ml PBS (140 mM NaCl, 2·7 mM KCl, 10 mM Na2HPO4, 1·8 mM KH2PO4, pH 7·3). Cells were broken open at 4 °C using a French press at 104 kPa (two passes), and the resulting extract centrifuged at 20 000 g for 30 min. The supernatant was collected and passed through a 2 ml Redi-Pack glutathione agarose bead column (Amersham Pharmacia), washed with 10 volumes of PBS buffer, and eluted with three 2 ml fractions of 50 mM Tris/HCl buffer, pH 8·0, containing 10 mM reduced glutathione. Fractions containing PrpR protein were dialysed against 10 mM Tris/HCl buffer, pH 7·9, containing 1 mM EDTA, 100 mM NaCl and 0·55 M glycerol. The PrpR protein was excised from the GST N-terminal fusion using thrombin protease. Excision from the tag leaves two amino acid residues from the GST protein tag in the N-terminus of PrpR (glycineserine). Protein released by this treatment was removed from solution by incubating the reaction with 0·1 ml of glutathione agarose beads. Thrombin cleavage of the GSTPrpR fusion protein was monitored by SDS-PAGE (Laemmli, 1970) and Coomassie Brilliant Blue staining (Sasse, 1991
). Purity of the cleaved protein was determined by SDS-PAGE and by passage through a sieving column. Purified protein was frozen in liquid nitrogen and stored at 80 °C until use.
Overproduction and purification of constitutive forms of PrpR protein.
Plasmids pPRP158, pPRP160 and pPRP161 were transformed into E. coli strain BL21 A-1 cells (Invitrogen) and transformants were selected for ampicillin resistance on LB agar medium containing ampicillin. An Apr transformant of each clone was used to inoculate LB medium (5 ml) containing ampicillin and 11 mM glucose. The cultures were grown overnight at 37 °C with shaking, and used to inoculate 500 ml of the same medium, which was incubated at 30 °C for 23 h until an OD650 of approximately 0·6 was reached. At this point, cultures were cooled to 15 °C, and expression of T7 RNA polymerase was induced with 13·5 mM L-(+)-arabinose and 1 mM IPTG. After induction of T7 RNA polymerase, cultures were incubated at 15 °C overnight, and cells were harvested by centrifugation at 8000 g at 4 °C for 15 min in a Sorvall RC5-B refrigerated centrifuge (Dupont Instruments) equipped with a GSA rotor. Cells were resuspended in 10 ml column buffer (20 mM HEPES, pH 8·0, containing 100 mM NaCl, 1 mM EDTA and 0·1 %, w/v, Triton X-100), and broken at 4 °C by passing them three times through a cell-disruption French press at 104 kPa. Cell-free extracts were treated with DNase I (5 µl, 2 mg ml1) for 10 min at room temperature and centrifuged for 20 min at 20 000 g, and the clarified supernatant was filtered through a 0·45 µm filter (Nalgene). Filtered extracts were loaded onto agarose/chitin columns (New England Biolabs) with a bed volume of 5 ml previously equilibrated with 10 volumes column buffer at a flow rate of 0·5 ml min1. After loading the cell extract, the columns were washed with 100 ml column buffer. The on-column self-cleavage reaction of the intein tag was started by washing the columns with two volumes of column buffer containing 50 mM DTT, followed by overnight incubation at 4 °C. Cleaved PrpR proteins were eluted from the column in the first 20 ml after additional column buffer with DTT was added. Eluted proteins were dialysed against two two-litre changes of 50 mM Tris/HCl buffer, pH 7·9, containing 100 mM NaCl, 1 mM EDTA, 0·1 mM DTT and 1·4 M glycerol. A third two-litre batch of buffer was used to dialyse proteins overnight at 4 °C. Proteins were frozen in pellets in the dialysis buffer using liquid nitrogen and stored at 80 °C until use. Purity of the protein preparations was determined by SDS-PAGE and by gel filtration FPLC analysis using a SuperdexTM 200 HR 10/30 column attached to an ÄKTA explorer (Amersham Pharmacia Biotech). The column was equilibrated and developed with 50 mM Tris/HCl buffer, pH 7·9, containing 100 mM NaCl and 1 mM EDTA. The column was developed at a flow rate of 0·7 ml min1; protein elution from the column was detected at A280. No significant contamination was detected in either of these analyses.
Assays
Electrophoretic mobility shift assay (EMSA).
To determine the DNA binding conditions for PrpR, an EMSA was performed. A 270 bp DNA fragment containing the intergenic region (IR) between the prpR and prpBCDE transcription units was amplified by PCR using primers R-IR (5'-CTG AAT TCG TGG GCA GTC GTC AT-3') and B-IR (5'-TAG GAT CCC GAA TGT AAA GAC AT-3'). This fragment was labelled with 5000 Ci mmol1 [-32P]ATP (ICN Biochemicals) using T4 polynucleotide kinase (Promega) following manufacturer's instructions, and purified using a Qiagen PCR purification kit. The specific activity of the radioactively labelled fragment was determined as described by Berger (1984)
. To determine the DNA concentration of the labelled probe, a PCR reaction was treated in parallel to the labelled PCR reaction (except for the addition of radioactive ATP) and then its DNA concentration determined by A260/280 to be 1 nmol ml1. The labelled probe was estimated to have the same concentration. The unlabelled probe was subsequently used in specific competition experiments. Reaction mixtures (20 µl) contained 20 mM Tris/HCl buffer, pH 7·4, 2 mM MgCl2, 1 mM CaCl2, 0·1 mM EDTA, 40 mM KCl, 100 µg BSA ml1, 20 µg salmon sperm DNA ml1, 20 000 c.p.m. of labelled probe, and various amounts of GST-fusion purified wild-type PrpR. When indicated, ATP and ADP were added to a final concentration of 1 mM. 2-MC was added to a final concentration of 5 µM. Bound complexes were separated in a non-denaturing 5 % polyacrylamide gel (25 mM Trizma base, 250 mM glycine buffer, pH 8·3), which was pre-run at 100 V for 30 min. The gel was dried onto filter paper and exposed to a fluorescent screen. Intensity of the signals was visualized using a STORM phosphorimager (Molecular Dynamics).
DNA footprinting
The sequence of the site to which PrpR binds was determined using a DNase I protection assay. For this purpose, assay mixtures with 20 000 c.p.m. of labelled DNA fragment and the indicated amounts of wild-type PrpR (derived from the GST-fusion protein) were incubated for 30 min at 37 °C under the same conditions as those of the EMSA assay. After this, 0·5 µg ml1 DNase I was added to the reactions, and the reactions were stopped exactly 2 min after the addition of DNase I with 180 µl of a solution containing 10 µg tRNA ml1 and 0·7 M ammonium acetate in 100 % ethanol. The reactions were kept in an ethanol/dry ice bath for 15 min, and then centrifuged for 15 min at 18 000 g. The supernatant was decanted and the pellet washed with 70 % ethanol. The pellet was dried under vacuum for 10 min and resuspended in DNA sample buffer (Promega). The samples were heated at 90 °C for 3045 s immediately before being loaded onto a 6 % denaturing polyacrylamide gel, which was run at 55 W. The gel was transferred onto filter paper, dried and exposed to a fluorescent screen for 16 h. Results were visualized using a STORM phosphorimager.
ATPase activity assay.
The wild-type form of PrpR (derived from both the GST fusion and CBPintein fusion) and the constitutive isoforms of PrpR (PrpRc, PrpRA162T and PrpRR24S, derived from the CBPintein fusion) were assayed for ATPase activity by measuring the release of radioactive orthophosphate from [-32P]ATP at 5000 Ci mmol1 (ICN Biochemicals). Assays were set up in a 60 µl volume of a reaction buffer containing 25 mM Tris/acetate buffer, pH 7·9, 8 mM magnesium acetate, 10 mM KCl, 1 mM DTT, 3·5 % PEG 6000 and 100 µg BSA ml1, the indicated amounts of a PCR-amplified fragment of DNA from the intergenic region between the prpR gene and the prpBCDE operon, and 2-MC. The ATP mix was prepared by adding 15 µCi of labelled ATP to 60 µl of a 20 mM solution of ATP. Purified protein was added to the reaction mixture to a final concentration of 1 µM and incubated for 5 min at 37 °C. The ATPase reaction was started upon the addition of 6 µl of [
32-P]ATP mix and incubated for 50 min. Ten microlitre samples were taken every 10 min and the reaction was stopped by the addition of 50 µl 1 M formic acid. Released orthophosphate was separated by TLC, spotting 5 µl on a polyethyleneimine cellulose plate (Merck), and developed with 0·4 M K2HPO4/0·7 M boric acid as the mobile phase. After this, the plate was air-dried and exposed overnight to a phosphor screen. Signal intensity was assessed in a Cyclone phosphorimager (Packard). Data are reported as the percentage of orthophosphate released per minute per mg of protein. The percentage of orthophosphate is defined as the amount of radioactivity at Rf=0·9 divided by the amount of radioactivity of [
-32P]ATP (Rf=0·5). Linear regression analysis of the data points for each assay condition showed a correlation coefficient greater than 0·9.
-Galactosidase activity assay.
Levels of -galactosidase enzyme activity were measured as described by Escalante-Semerena & Roth (1987)
. A unit of activity is defined as the amount of enzyme needed to cleave one nmol of ONPG per minute per OD650 unit. Activation of the PprpBCDE promoter as a function of 2-methylcitric acid (94 % purity, CDN Isotopes) (Busch et al., 2002
) was performed in strain JE2170 (prpC114 : : MudJ). The latter was grown overnight in 2 ml LB/kanamycin, and 20 µl of this culture was used to inoculate 2 ml NCE minimal medium supplemented with glycerol and kanamycin. The culture was incubated with shaking at 37 °C until an OD650 of 0·50·6 was reached. At that cell density, 2-MC was added to a final concentration of 1, 10 or 20 mM, and the cultures were incubated for 30 min.
-Galactosidase activity was measured in 0·5 ml samples.
In vitro transcription assays.
Plasmid pPRP164 was purified and cleaned with an equal volume of phenol/chloroform (1 : 1), followed by ethanol precipitation. This plasmid produced a transcript of approximately 135 nt. E. coli-derived RNA polymerase and 54 were gifts from R. Burgess, and IHF from M. Filutowicz. For this assay, CBPintein derived PrpR, PrpRc PrpRA162T and PrpRR24S were used. Assays were carried out at 37 °C in 10 µl reactions containing the indicated amount of activator with 20 nM DNA template, 25 nM RNA polymerase core enzyme, 100 nM
54 protein, 4 mM ATP, 40 nM IHF protein, in Tris/acetate buffer (25 mM Tris/acetate, pH 7·9, 8 mM magnesium acetate, 10 mM KCl, 1 mM DTT and 3·5 % PEG 6000) (Chaney & Buck, 1999
). Assays were preincubated at 37 °C for 20 min. Reactions were started by adding a mixture containing 0·4 mM ATP, CTP and GTP, 0·05 mM UTP and 1·5 µCi [
-32P]UTP, incubated for 20 min and stopped with an equal volume of formamide stop buffer (95 % formamide, 20 mM EDTA, pH 8·0, 0·05 % bromophenol blue and 0·05 %, w/v, xylene cyanol). Ten microlitres were loaded onto a 6 % denaturing polyacrylamide gel and run at 200 V. The gel was dried, exposed to a phosphorimager screen overnight and transcripts were detected using a STORM phosphorimager. The intensity of each band was quantified and normalized against the background using ImageQuant analysis software (Molecular Dynamics). One transcription arbitrary unit is defined as 1x103 band intensity units.
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RESULTS |
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To determine whether 2-MC was required for the transcriptional activation of the prpBCDE genes or not, co-activator-dependent transcription of the prpB promoter by PrpR was tested in vitro. Increasing amounts of 2-MC in the reaction mixture correlated with increased transcription of PrpR (Fig. 3). The effect of 2-MC displayed saturation kinetics, with little change in the amount of transcript at 2-MC concentrations higher than 2·5 µM (data not shown). Control reactions using a
70 rRNA promoter (plasmid p770) and the constitutive isoform of PrpR, PrpRc (Palacios & Escalante-Semerena, 2000
) indicated that 2-MC by itself did not have a negative effect on transcription. Propionate or citrate failed to substitute for 2-MC in the in vitro transcription assays (data not shown). The amount of 2-MC required to activate transcription in vivo was significantly higher than that required for activation in vitro. The simplest explanation for this observation may be that the cell has difficulty transporting 2-MC into the cytoplasm.
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Effect of 2-MC.
The ATPase specific activity of wild-type PrpR and of PrpRA162T and PrpRR24S mutant proteins was determined as a function of the concentration of 2-MC in the reaction mixture. The ATPase activity of the wild-type protein in the absence of 2-MC was unaffected by the presence of 2-MC over a three-orders-of-magnitude concentration range (0·001 to 1 mM): 72 % liberated PO4 min1 in the presence of 1 µM 2-MC; 56 % in the presence of 1 mM 2-MC. Specific activities with 2-MC at 0·005, 0·010, 0·1 and 0·5 mM 2-MC were 67±5 % liberated PO4 min1. The ATPase specific activity of co-activator-independent point mutants (which are discussed further) PrpRA162T (76±8) and PrpRR24S (66±6) was not significantly different than that of the PrpRc isoform.
Effect of DNA.
We determined the effect of DNA on the ATPase activity of PrpR. The ATPase specific activity of the constitutive isoform PrpRc did not change when DNA containing the PrpR binding site was added to the reaction mixture (specific activity was 237, 254, 257 and 253 % liberated PO4 min1 at 0, 0·1, 0·2 and 1·0 µM DNA, respectively). Similarly, the specific activity of the wild-type PrpR protein (68 %) did not change as a function of the concentration of DNA in the reaction mixture (54 % with 1 µM DNA in the reaction mixture).
Effect of other factors.
Addition of RNA polymerase, 54 and IHF to the reaction mixture did not affect the ATPase activity of any of the proteins tested (data not shown). Two mutant PrpR proteins (PrpRA162T, PrpRR24S) had ATPase activity equivalent to that of the wild-type PrpR protein, and their ATPase activity was not affected by 2-MC (data not shown).
The PrpR binding site
The DNA-binding properties of the PrpR protein were investigated using an electrophoretic mobility shift assay. PrpR protein bound to the DNA region between the prpBCDE operon and prpR (Figs 1a and 4a). These data and those obtained with control experiments (Fig. 4
b) indicated that binding of PrpR to the DNA used in the assay was specific. The three-band pattern observed in the lanes where specific competitor DNA was added may correspond to different degrees of oligomerization of PrpR (i.e. bound dimer, tetramer, hexamer) (Fig. 4b
). Results from DNase I protection experiments indicated that the PrpR binding site spanned nucleotides 110 to 145 relative to the PprpBCDE promoter (Fig. 5
). Analysis of the protection pattern showed that this region contained two sites with dyad symmetry. Protection of the binding site correlated with the amount of PrpR in the mixture. PrpR binding sites were defined by a region of strong DNase I hypersensitivity located at the 3' end of the symmetric regions (Fig. 5a
). PrpR bound to two regions which have the consensus sequence 5'-CGTTTCATGAAACG-3' and which span from 110 to 123 and from 132 to 145 bases from the 24 element of the prpB promoter (Fig. 5b
). The consensus sequence can be generated by a twofold rotation of the half site 5'-CGTTTCA-3', suggesting that the site is recognized as an inverted repeat. Addition of 2-MC (10 µM), ATP or ADP (1 mM) to the reaction mixture did not have any discernible effect on the binding pattern in either EMSA or DNase protection assays (data not shown).
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DISCUSSION |
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Why is 2-MC, not propionyl-CoA, the co-activator of PrpR?
This question might be best answered within the context of propionate toxicity. Because 2-MC is a potent inhibitor of cell growth (Horswill et al., 2001), it makes sense for the cell to have a mechanism that would sense 2-MC and immediately trigger the synthesis of the enzymes that degrade it. If propionyl-CoA were toxic to S. enterica, one would expect the primary pathway for detoxification (the 2-MC cycle) to be readily induced. In fact, the prpBCDE operon is not induced by strains proficient in the synthesis of propionyl-CoA but deficient in the synthesis of 2-MC (Tsang et al., 1998
). Similar induction of catabolic genes by a toxic metabolic intermediate has been observed in the
-ketoadipate pathway, in which cis,cis-muconate induces the genes that catalyse the conversion of benzoate to tricarboxylic acid cycle intermediates (Collier et al., 1998
).
Interactions of PrpR with its DNA binding site
The DNA sequence recognized by PrpR is composed of two sites, which were termed proximal (P) and distal (D) to reflect their location relative to the prpB promoter (Fig. 5b). The differences between the two sites may result in different affinities of PrpR for each site, which would explain the multiple-band pattern observed in the gel-shift experiments (Fig. 4
), the slightly weaker protection pattern observed for the distal site in the footprinting experiment, and the appearance of a hypersensitive site 3' to the distal site only when concentrations of PrpR above 5 pM were used (Fig. 5a
). Previous studies have shown that NtrC, a widely studied member of this family of activators, binds to its ELE and activates transcription through the assembly of large oligomers in which some of the dimers are not in direct contact with the DNA (Wyman et al., 1997
). These oligomers appear to form a hexameric ring structure, which is also a feature of the AAA+ superfamily of proteins (Vale, 2000
; Zhang et al., 2002
). The multiple-band pattern observed in the EMSA may be the result of PrpR complexes of different oligomerization state binding to the two ELE sites. The overlap of the D site with the 35 element of the prpR promoter suggests that PrpR may affect its own synthesis (Fig. 5b
). Further testing is needed to confirm these ideas.
The lack of an effect by 2-MC or ATP/ADP on the ability of PrpR to bind DNA was surprising because of the known stimulatory effect of the co-activator and ATP on DNA binding by other 54-dependent transcriptional activators (Austin & Dixon, 1992
; Lee et al., 1994
). It is possible that PrpR represents a case in which the binding of 2-MC affects other aspects of the activation process. Alternatively, other factors involved in vivo may have been missing in the in vitro experiments. The former possibility is discussed further.
Role of 2-MC
The signal-sensing domain of PrpR has a negative effect on the activity of the protein, since removal of the domain yields a constitutively active protein. Removal of the sensing domain has similar effects on the activity of other members of this family of activators (Fernandez et al., 1995; Lee et al., 1994
; O'Neill et al., 1998
). In other
54 activators, binding of the co-activator molecule or phosphorylation of the sensing domain triggers oligomerization, which is further enhanced by nucleotide and DNA binding. This set of events increases the activator's ATPase activity, stabilizes the oligomeric form of the activator protein and results in productive interactions with RNA polymerase, thus activating transcription (Austin & Dixon, 1992
; O'Neill et al., 1998
; Weiss et al., 1991
; Zhang et al., 2002
).
We have no evidence that PrpR fits this model. In fact, under the conditions tested, the ATPase activity in the wild-type PrpR protein was not stimulated by addition of 2-MC over a broad range of concentrations (0·0011 mM). The fact that a single amino acid change (PrpRA162T) can completely bypass the need for 2-MC without affecting the ATPase activity of the protein indicates that the effect of 2-MC binding on PrpR-dependent activation of prpBCDE expression is not due to an increase in the ATPase activity of the protein. It appears that the highest level of ATPase activity in PrpR can only be attained by removal of the entire sensing domain (PrpRc). The ATPase activity in the PrpRc protein, however, is only 2·5-fold greater than that of the wild-type protein. It is clear that the ATPase activity of the constitutive protein PrpRc is not stimulated by addition of DNA. It is possible that ATP binding, hydrolysis, oligomerization and DNA binding are unregulated events in PrpR, and 2-MC binding may expose the GAFTGA motif, a highly conserved region in the catalytic domain found to be essential for interaction with RNA polymerase/ 54 and for transduction of the ATP hydrolysis signal (Lee et al., 2003
). PrpR, however, contains an alanineserine substitution in this motif (GAFTGS) that does not affect the transcriptional activity of the protein (Palacios & Escalante-Semerena, 2000
). A more detailed analysis of this and other structural differences is needed, and would provide important information to understand the activation mechanism of PrpR.
PrpR is different from other well-studied members of this family of activators, such as XylR and DmpR, in which the region linking the sensing and catalytic domains contains a coiled-coil structure that is important for controlling binding of the effector molecule and activation of the protein (Garmendia & de Lorenzo, 2000; O'Neill et al., 2001
). A coiled-coil structure was not detected in several other members of this family of activators that sense small-molecule effectors, including PrpR. When the primary amino acid sequence of PrpR was analysed using the COILS coiled-coil structure algorithm (Lupas et al., 1991
), no coiled-coil structures were predicted in PrpR (data not shown).
We note that none of the mutations that led to 2-MC-independent PrpR activity were mapped in the linker region between the A and C domains (Table 2). We propose that PrpR and other members of this family of regulators that sense small-molecule co-activators and lack this coiled-coil motif constitute a subclass which uses a different mechanism for signal propagation within the protein. Structural studies of the wild-type and constitutively active mutant proteins may provide insights into the signal-transduction-pathway signal in PrpR.
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ACKNOWLEDGEMENTS |
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Received 4 May 2004;
revised 12 July 2004;
accepted 17 August 2004.
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