| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene
asz
yna Konopa
Department of Molecular Biology, University of Gda
sk, Kadki 24, 80-822 Gdask, Poland
Correspondence
Alina Taylor
ataylor{at}biotech.ug.gda.pl
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
54 promoter in the regulatory region of the rpoH gene, described previously, is a functional promoter, P6. The evidence for this conclusion is: (i) the specific binding of the 54–RNAP holoenzyme to P6, (ii) the location of the transcription start site at the predicted position (C, 30 nt upstream of ATG) and (iii) the dependence of transcription on 54 and on an ATP-dependent activator. Nitrogen starvation, heat shock, ethanol and CCCP treatment did not activate transcription from P6 under the conditions examined. Two activators of 54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspF
HTH, a derivative of PspF, devoid of DNA binding capability but retaining its ATPase activity, was used for transcription in vitro, taking advantage of the relaxed specificity of ATP-dependent activators acting in solution. In experiments in vivo overexpression of PspFHTH from a plasmid was employed. Thus, the 54-dependent transcription capability of the P6 promoter was demonstrated both in vivo and in vitro, although the specific conditions inducing initiation of the transcription remain to be elucidated. The results clearly indicate that the closed 54–RNAP–promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
32 (Arsène et al., 2000
; Zhao et al., 2005) and 24 RNA polymerase (RNAP) subunits (Erickson & Gross, 1989; Connolly et al., 1999), the rpoH and rpoE gene products, respectively. A third regulon of the stress response has been identified from recent data. It is under the control of the 54–RNAP subunit (the rpoN gene product). The first 54-dependent heat shock operon, pspA–E, was described by Model's group (Brissette et al., 1991; Model et al., 1997). Later, it was found that the ibpB gene is transcribed not only from the 32 promoter upstream of the ibpAB operon (Allen et al., 1992), but also from the internal 54 promoter situated in the ibpA–ibpB intergenic space (Kuczyska-Wi
nik et al., 2001). Moreover, a canonical sequence of a 54 promoter, proximal to the rpoH coding sequence, was found by sequence analysis (Pallen, 1999). Information as to whether the sequence represents a functional promoter is currently lacking.
Transcription of the rpoH gene can be initiated from four previously described promoters (Fig. 1). Three of them, P1, P4 and P5, are recognized by 70–RNAP (Erickson et al., 1987; Nagai et al., 1990; Ramirez-Santos et al., 2001; Solis-Guzman et al., 2001) and P3 is recognized by 24–RNAP (Erickson & Gross, 1989; Wang & Kaguni, 1989a; Missiakas & Raina, 1998). The P2 promoter was found only in one E. coli strain, SC122 (Erickson et al., 1987), and was absent from MG1655. Transcription of the rpoH gene from these promoters is regulated by CRP–cAMP, CytR (Ramirez-Santos et al., 2001; Kallipolitis & Valentin-Hansen, 1998) and DnaA (Wang & Kaguni, 1989b; Messer & Weigel, 1997) proteins.
|
54 promoter of the rpoH gene is functional, it should mean that stress responses encompass as yet unidentified processes. Functional 54 promoters are rare in the E. coli genome: their number was estimated to be about 30, though sequences resembling canonical 54 promoters are abundant (Reitzer & Schneider, 2001). The 54 subunit is not essential for E. coli growth under laboratory conditions; however, it is necessary for expression of genes involved in diverse processes like nitrogen metabolism, transport of dicarboxylic acids, pilus formation, formate dehydrogenase synthesis, metabolism of aromatic compounds (xylene, toluene), and expression of the pspA–E operon and of the ibpB gene encoding a small heat shock protein.
E. coli cells contain seven different subunits (Arsène et al., 2000; Wösten, 1998) that form two classes characterized by sequence homology and specificity of promoter recognition. Six of them belong to class I, represented by 70. The 54 subunit is a unique member of class II (Merrick, 1993; Shingler, 1996; Wösten, 1998), sharing no homology with class I. The 54 subunit recognizes the conserved sequence YTGGCACG-N4-TTGCWNN of the promoters located at –12 and –24 bp from the transcription start site (Barrios et al., 1999). Initiation of 54–RNAP-dependent transcription resembles eukaryotic transcription initiation by RNAP II. (Guo et al., 1999, 2000; Fu et al., 2000). A closed complex of class I–RNAP–promoter isomerizes spontaneously to the active, open complex. In contrast, isomerization of the 54–RNAP–promoter closed complex requires interaction with the specific enhancer-bound, ATP-dependent activator (reviewed by Merrick, 1993; Shingler, 1996). Contact between the activator and the 54–RNAP–promoter complex is achieved by DNA looping, facilitated either by the integration host factor (IHF) protein or by intrinsic DNA topology (Pérez-Martin et al., 1994; Carmona et al., 1997). Control, imposed on the DNA melting step, requires ATP hydrolysis and involves the promoter –12/–11 element (Guo et al., 1999, 2000) bound inside the RNAP channel (Polyakov et al., 1995; Zhang et al., 1999; Severinov, 2000; Foster et al., 2001).
Activators of 54-dependent promoters have a modular structure and share conserved sequence motifs with the AAA+ protein family (Shingler, 1996; Zhang et al., 2002; Cannon et al., 2003; Studholme & Dixon, 2003). The N-terminal domains of the majority of these activators act as specific sensors of inducing signals. NtrC may be regarded as a representative of this group. In response to nitrogen limitation, the ATPase activity of NtrC is positively regulated by phosphorylation of its N-terminal domain by the NtrB kinase and stimulated by DNA binding (Weiss et al., 1991, 1992).
Members of another smaller, group of activators of 54-dependent promoters, which lack the N-terminal regulatory domain, include PspF of E. coli (Jovanovic et al., 1996; Model et al., 1997) and HrpR of Pseudomonas syringae (Grimm et al., 1995). PspF, the activator of the pspA–E operon, occurs in cells at low concentrations and is constitutively active. Modulation of its activity, unlike that of NtrC, depends on protein regulators: PspA (negative) or PspB and PspC (positive) (Shingler, 1996; Elderkin et al., 2002; Hankamer et al., 2004). PspF responds to heat shock and ethanol treatment, but the strongest reaction is evoked by bacterial membrane impairments coincident with the dissipation of charge as by the gene IV protein of filamentous phages (e.g. f1, M13) or by carbonylcyanide m-chlorophenylhydrazone (CCCP; Model et al., 1997). The molecular mechanisms of signal reception and transduction in this system are not yet understood.
The purpose of this work was to establish whether the 54–RNAP holoenzyme binds the putative 54-dependent promoter (P6) in the regulatory region of the rpoH gene, and whether it is able to initiate transcription from P6 in vitro and in vivo. We present evidence of the holoenzyme binding and initiation of transcription in vitro and in vivo, based on gratuitous activation by PspFHTH. The identity of the specific activator and inducing conditions remain to be elucidated.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The bacteria were grown in Luria–Bertani (LB) medium at 30 °C (unless otherwise stated). When appropriate the following antibiotics were added: ampicillin (100 µg ml–1), tetracycline (15 µg ml–1), kanamycin (30 µg ml–1) and chloramphenicol (34 µg ml–1). Transformation of E. coli cells was performed according to Sambrook et al. (1989) by using the CaCl2 procedure.
|
) were amplified by PCR with the corresponding primers.
Proteins.
The His-tagged proteins listed below were purified by Ni-affinity chromatography using the BioLogic LP chromatography system (Bio-Rad). His–54 and His–NtrC proteins were overproduced from strains BL21(DE3)(pS54-2) and BL21(DE3)(pNTRC-3), respectively and purified as described by Rippe et al. (1997, 1998). His–PspF and His–PspFHTH were obtained by overproduction from SG13009(pREP,pMJ16) and K1527 (pMJ15) respectively and purified according to Jovanovic et al. (1999). The IHF protein was purified as described by Filutowicz et al. (1994) after overexpression from strain DH5
(pHN
). 70 was overproduced from CF1690lac(pVI690) and purified as described by Fujita & Ishihama (1996). E. coli core RNAP was purchased from Epicentre Technologies. Reconstituted RNAP holoenzyme was obtained by incubating the core RNAP with an appropriate subunit at a 1 : 4 molar ratio for 15 min at 30 °C in STA buffer (25 mM Tris/acetate pH 8.0, 8 mM magnesium acetate, 10 mM KCl, 1 mM DTT, 3.5 % PEG). NtrC was phosphorylated to NtrC-P before use by the addition of carbamyl phosphate (10 mM) to the reaction mixture.
Electrophoretic mobility shift assays (EMSAs).
The DNA fragments W1-W2, W2-W5 and W8-W9 were end-labelled with [
-32P]ATP (166.5 TBq mmol–1) by using T4 polynucleotide kinase (Promega). DNA–protein binding was carried out in STA buffer supplemented with BSA (100 µg ml–1) and poly[dI–dC] (30 µg ml–1) for 20 min at 30 °C and was stopped by the addition of 2 µl loading dye (0.05 % bromophenol blue, 50 %, v/v, glycerol). The amounts of DNA and holoenzyme are given in the legends for Figs 2–5. Electrophoresis was performed in 4.5 % polyacrylamide gel in Tris/glycine buffer (25 mM Tris, 200 mM glycine) at 8 V cm–1 for 2 h. The gels were dried and submitted to autoradiography or analysed with a Molecular Imager FX (Bio-Rad).
|
|
|
|
). The W1-W2 (707 bp) DNA fragment containing the putative P6 promoter (200 nM) and 54–RNAP (35 nM) were incubated for 15 min at 30 °C in a final volume of 20 µl. For visualization of PspF or NtrC-P binding the W1-W2 fragment was prepared by PCR with biotinylated W2 primer (W2biot). W1-W2biot (620 ng) was incubated with streptavidin (Promega) in a total volume of 40 µl in TM buffer for 1 h at 37 °C. The excess of streptavidin was removed by filtration through Amicon Microcon-PCR (Milipore) filters. The PspF or NtrC-P proteins (500 nM) and the W1-W2biot DNA fragment (200 nM) were incubated at 30 °C for 15 min in buffer A.
The reaction products were cross-linked by the addition of 0.2 % glutaraldehyde, and, after incubation at room temperature for 15 min, diluted 1 : 20 with TM buffer. Preparations for electron microscopy were made by adsorption to mica (Spiess & Lurz, 1988), stained with uranyl acetate, platinum–carbon coated and analysed with a Philips CM100 electron microscope. The lengths of the DNA fragments and the positions of complexes were measured by using an electronic digitizer and evaluated with a computer program as described previously (Weigel et al., 1997).
DNase I footprinting.
Primers were end-labelled with [-32P]ATP (166.5 TBq mmol–1) and T4 polynucleotide kinase (Promega). DNA fragments were PCR amplified using 32P end-labelled W7 primer and unlabelled W3 primer for 54, 54–RNAP and 70–RNAP footprints. 32P end-labelled W9 primer and unlabelled W8 primer were used for IHF footprints. DNase I footprinting assays were performed as described by Leblanc & Moss (2001) in a total volume of 10 µl of STA buffer. The labelled DNA fragments at a final concentration of 10 nM were incubated, for 15 min at 30 °C, with proteins in quantities given in the figure descriptions. DNase I (5x10–4 units) was added and after incubation for 1 min at 37 °C the reaction was stopped by the addition of EDTA to a final concentration of 50 mM followed by heating for 2 min at 95 °C in formamide loading dye. The reaction products were separated on an 8 % PAGE/urea gel. In parallel, the products of DNA sequencing reactions, performed using the fmol DNA Cycle Sequencing System (Promega), were separated. Gels were subjected to autoradiography or exposed to Kodak screen and analysed using the Molecular Imager FX (Bio-Rad).
In vitro transcription.
Multiple-round transcription assays were performed essentially as described by Wigneshweraraj et al. (2003) with minor modifications. Briefly, the supercoiled DNA template pTE103-W1-W2 for in vitro transcription was prepared by cloning the W1-W2 (707 bp) DNA fragment digested with BamHI and EcoRI into the pTE103 transcription vector, purified by centrifugation (at 2x105 g, for 16 h) through a caesium chloride/ethidium bromide gradient and verified by sequencing (Macrogen). Reactions were conducted in STA buffer supplemented with BSA (100 µg ml–1), in a total volume of 20 µl. First, 1 µg of template DNA was incubated for 10 min at 37 °C with 100 nM 54–RNAP and each of the activators (PspF, PspFHTH or NtrC-P) at concentrations indicated in the figure legends. Then ATP was added to the final concentration of 5 mM, and the reaction was continued for 5 min to allow for izomerization of the closed complex into an active, open complex. Transcription was initiated by the addition of ribonucleotide mixture (0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 0.06 mM UTP, 0.11-0.185 MBq [-32P]UTP) and incubation for 10 min at 37 °C. Then heparin (100 µg ml–1) was added and incubation was continued for 5 min. The reaction was stopped by the addition of a formamide loading dye. Samples were heated (2 min at 95 °C) and subjected to electrophoresis in 4.5 % PAGE/urea gel at 250 V in TBE buffer. Gels were subjected to autoradiography or exposed to Kodak screen and analysed using the Molecular Imager FX (Bio-Rad).
Primer extension analysis.
Cells were grown in LB medium at 30 °C, to an OD575 of 0.4 and harvested by centrifugation at 2x103 g for 10 min. Total RNA was isolated using the RNA Prep Plus kit (A & A Biotechnology). For PspFHTH or 54 overexpression from pMJ13 or pVI688, respectively, cultures at an OD575 of 0.4 were induced by the addition of IPTG (1 mM) and incubation was continued for 0.5–1 h for protein synthesis. The W7 primer complementary to nucleotides +73 to +44 of the rpoH gene was 5'-end labelled using T4 polynucleotide kinase (Promega) and [-32P]ATP (166.5 TBq mmol–1). RNA (50 µg) was mixed with the labelled W7 primer (1 pmol) and heated to 85 °C for 20 min. Then M-MuLV reverse transcriptase buffer was added and the W7 primer was hybridized to the RNA at 62 °C for 1 h. Then, RevertAid H Minus M-MuLV reverse transcriptase (Fermentas), ribonuclease inhibitor (1 unit µl–1) and dNTPs (2.5 mM each) were added. Primer extension reactions were carried out at 42 °C for 1 h and stopped by adding loading dye containing formamide. DNA sequencing reactions were carried out with the same primer using the fmol DNA Cycle Sequencing System (Promega). Reaction products were heated for 2 min at 95 °C and loaded on a 8 % PAGE/urea gel together with products of the DNA sequencing reactions carried out with the fmol DNA Cycle Sequencing System. Gels were subjected to autoradiography or exposed to Kodak screen and analysed using a Molecular Imager FX (Bio-Rad).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
54 promoter in the promoter region of the rpoH gene was known (Pallen, 1999), raising the question whether it might be an active promoter. Analysis of the upstream sequence of the rpoH gene by a BLAST search showed that the 54 promoter was correctly situated according to the rules proposed by Reitzer & Schneider (2001) for active 54 promoters. Moreover, an IHF–binding site homologous to the consensus sequence was found at the position –349 to –362 (Fig. 1b) (Craig & Nash, 1984; Goodrich et al., 1990). The search for a possible enhancer was focused on sequences resembling those of PspF- and NtrC-binding sites, since PspF is the activator of the 54-dependent stress-induced pspA–E operon, and NtrC activates a few 54-dependent promoters. It seemed probable that one of these activators could participate in transcription of the rpoH gene encoding 32, the main stress-response factor. Sequence analysis of the rpoH regulatory region indicated the presence of some imperfect NtrC- binding sites and PspF-binding sites spread over the whole region of interest (Fig. 1b).
54–RNAP binding to the putative P6 promoter
54–RNAP binding to P6 was tested by electrophoretic mobility-shift assay (EMSA), electron microscopy (EM) and DNase I footprinting. For EMSA the 277 bp DNA fragment containing the putative P6 promoter was prepared by PCR using primers W2 and W5 (Fig. 1a) and end-labelled with [-32P]ATP. We observed a DNA shift at a 54–RNAP : DNA molar ratio of 5 : 1 or higher (Fig. 2a), a preliminary indication of 54 holoenzyme binding to this DNA region. EM gave strong evidence for specific binding of the holoenzyme to the putative 54 promoter, P6 (Fig. 2b). The 707 bp DNA fragment W1-W2 (encompassing W2-W5) was used. Statistical analysis of approximately 100 EM images revealed that 54–RNAP binding was centred on the postulated promoter (Fig. 2c). The precise site of 54–RNAP binding was determined by DNase I footprinting (Fig. 3a). The W3-W7 (326 bp) -32P end-labelled DNA fragment was prepared by PCR, as detailed in Methods. In agreement with the EM results (Fig. 2b, c) 54–RNAP bound exactly at the postulated P6 promoter, covering the sequence –37 to –68 (Fig. 3). As a reference, DNase I footprinting with 70–RNAP was performed. The protected region from –72 to –117 corresponded to the P5 and P4 promoters. The nucleotide sequence of the protected area is shown below the autoradiogram (Fig. 3b). There was no binding of the 54 protein alone, which is observed only with 54 promoters containing a stretch of four Ts immediately upstream of the conserved GC of the promoter sequence (Buck & Cannon, 1992). P6 contains a TTGTT sequence instead. A DNA fragment with no protein added (Fig. 3a, lane 1) and a DNA fragment incubated with core RNAP only (lane 13) were used as controls and showed negative results as expected.
IHF, NtrC and PspF binding upstream of the P6 promoter
Analysis of the regulatory region of the rpoH gene revealed the presence of a putative IHF-binding site, with remarkable homology to the consensus site (Craig & Nash, 1984; Goodrich et al., 1990). To our knowledge this has never been detected before. To determine whether this site actually binds IHF, EMSA and DNase I footprinting were performed. EMSA demonstrated IHF binding to the 277 bp DNA fragment W8-W9. Binding occurred at a molar ratio of IHF to DNA of 2 : 1 (Fig. 4a). DNase I footprinting showed IHF binding at the predicted site, i.e. at –337 to –383, corresponding to the extended IHF-binding site (Goodrich et al., 1990) (Fig. 1 and Fig. 4b, c).
The search for NtrC- or PspF-binding sites by sequence analysis of the 707 bp W1-W2 DNA fragment resulted in a complicated picture of several imperfect binding sites for each of these proteins (Fig. 1b). Nevertheless, it seems possible that some of them act as enhancers, binding the activator protein.
EMSA, EM and footprinting experiments were used to check the possible binding of PspF and NtrC-P (NtrC phosphorylated in vitro by carbamyl phosphate) to the 707 bp DNA fragment W1-W2. A high molar ratio of either of the two proteins (200 nM PspF monomer or 250 nM NtrC-P monomer per 2 nM DNA) was needed to produce a mobility shift (Fig. 5a, d). These data suggested that both activators bind DNA nonspecifically. For EM the 707 bp DNA fragment was end-labelled with streptavidin (see Methods), which served as a reference point for measurements of the position of the nucleoprotein complex. It was determined from the photomicrographs and by statistical analysis that both proteins, PspF and NtrC-P, bound all along the DNA fragment (Fig. 5b, c, e, f). Since the previously observed specific binding of the 54–RNAP detected by EM was limited to the narrow area of the P6 promoter, it was evident that neither of the activators bound specifically. Also, the results of NtrC-P and PspF DNase I footprinting were negative (data not shown). This evidence supports the hypothesis that PspF and NtrC-P are not the specific activators for the transcription from P6.
Transcription in vitro from the P6 promoter
The P6 promoter was tested for in vitro transcriptional activity in multiple-round transcription. The supercoiled template was prepared by cloning the W1-W2 DNA fragment containing P1, P3, P4, P5 and the putative P6 promoters in the pTE103 transcription vector (Elliott & Geiduschek, 1984). Each reaction mixture contained template DNA (pTE103-W1-W2), 54–RNAP and the activator protein at increasing concentration. Use was made of the relaxed specificity of activators on supercoiled templates (Dworkin et al., 1998), and PspF, NtrC-P and PspFHTH were employed. Each of these activators caused the initiation of transcription (Fig. 6a, b, c). PspF and NtrC-P stimulated transcription at much lower protein levels (about 50 nM) than PspFHTH (1 µM), perhaps because PspFHTH not only does not bind DNA, but also has ATPase activity 30-fold lower than PspF (Jovanovic et al., 1999). We concluded that 54–RNAP recognized the P6 promoter and formed a closed initiation complex capable of starting transcription in vitro provided that any of the ATP-dependent activators was present. Transcription in vitro (activated by PspF) was slightly inhibited by IHF (data not shown), which is consistent with the observation that IHF stimulates transcription only with a specific activator (Dworkin et al.1998).
|
HTH from the pspF877 gene, inducible by IPTG, and pREP, providing the lacI repressor. The specific conditions that would induce transcription from P6 were not known; however, use was made of the observation of Jovanovic et al. (1996) that overproduction of PspFHTH activated pspA operon expression even in the absence of inducing stimuli. P6 transcription appeared totally dependent on the overproduction of PspFHTH from pMJ13 induced by IPTG (Fig. 7a, b). The 5' end of the P6 transcript was found at position –30, from the rpoH start codon (+1). There was rough proportionality between PspFHTH concentration (dependent on the length of the induction time) and the level of the transcript (Fig. 7a). This transcription was abolished by the rpoN mutation (Fig. 7b, lane 9), though PspFHTH was overproduced to the same level in the wt and mutant strains, as confirmed by SDS–PAGE (data not shown). Complementation of the rpoN mutation by plasmid pVI688 overproducing 54 restored transcription from P6 (Fig. 7c), if PspFHTH was overproduced simultaneously with the factor. These results indicate that the transcription from P6 is 54-dependent and that the 54–RNAP is able to direct transcription of the rpoH gene from the P6 promoter if an activator is supplied.
|
). Also, induction of NtrC activity by nitrogen starvation was tested. None of these conditions activated the P6 promoter, as determined by primer extension. The results are consistent with neither PspF nor NtrC being a specific activator of this promoter. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
54-dependent P6 promoter in the regulatory region of the rpoH gene of E. coli is able to direct transcription. Binding of the 54–RNAP holoenzyme to the P6 promoter sequence was demonstrated by EMSA, electron microscopy and DNase I footprinting. The closed complex of the 54–RNAP and P6 promoter extended from –7 to –38 bp, relative to the transcription start site (–37 to –68 bp relative to the ATG). This is consistent with the size of the DNA area protected by closed complexes observed for other 54 promoters (Buck & Cannon, 1992; Wang et al., 1998). The closed complexes of these promoters require interaction with activators of the AAA+ protein type bound to remote enhancers. IHF is a frequent participant in the process. The presence of an IHF-binding site that corresponded to the consensus sequence (Craig & Nash, 1984; Goodrich et al., 1990) and demonstration of binding to IHF, was consistent with previous findings regarding the structure and functioning of most of the 54 promoter regulatory regions. The dependence of in vitro transcription on the AAA+ activators NtrC-P, PspF or PspFHTH (Fig. 6) was shown. Also an analogous situation produced in vivo by overproduction of PspFHTH, which does not bind DNA, from pMJ13 in E. coli cells resulted in activation of transcription from P6.
The location of the transcriptional start site 30 nucleotides upstream of the rpoH coding sequence by primer extension experiments (Fig. 7a, b, c) was in agreement with previous prediction (31 nt upstream according to Pallen, 1999). The start occurred in the statistically determined area for starts from the 54 promoters, i.e. between 8 and 21 nt downstream of the promoter's conserved GC (Barrios et al., 1999). The rpoN mutation abolished transcription from P6 as expected (Fig. 7b, lane 9).
We interpret these data, taken together, as evidence that 54–RNAP binds the P6 promoter and forms a closed complex able to initiate rpoH gene transcription in vitro and in vivo, provided that an ATP-dependent activator is present. However, the specific activator and conditions inducing transcription from the P6 promoter remain unknown. This makes assessment of the physiological importance of P6 difficult, but evolutionary conservation of the P6 sequence (Pallen, 1999) speaks for its usefulness in as yet unrecognized conditions.
The P6 promoter influenced transcription starting from the upstream promoters (P1–P5) of the rpoH gene. Formation of the stable, closed complex of 54–RNAP at the P6 promoter can be a spatial hindrance for the RNAP holoenzyme. Such a complex cannot be formed in the rpoN strain. Accordingly, an increased level of the transcription from the upstream promoters in the rpoN cells was observed (Fig. 7b, lanes 8 and 9) but not further explored. The sigma competition for the core RNAP, mediated by the alarmone ppGpp (Sze & Shingler, 1999; Maeda et al., 2000; Jishage et al., 2002; Laurie et al., 2003; Nyström, 2004; Magnusson et al., 2005) could also play an important role in the complicated regulation of rpoH transcription. Since core RNAP is limiting for transcription, the amount of 70 bound to RNAP is increased in the absence of 54.
In a paper by Reitzer & Schneider (2001), the authors mentioned unpublished results of experiments in which it was not possible to demonstrate the existence of a transcript from the P6 promoter in nitrogen-limited cells, which meant NtrC was not the activator of this transcription. We confirm this opinion. These results are also consistent with the negative result of our in vitro studies on PspF and NtrC-P binding to the rpoH regulatory region (Fig. 5).
There is a possibility that the unidentified enhancer for the activator may be found further upstream or downstream of the 707 bp (W1-W2) region tested. The enhancers binding ATP-dependent activators for the 54 promoters are usually situated about 100–160 bp upstream of the coding region; they may rarely be found 700 bp apart, but a larger distance is not excluded (Gralla & Collado-Vides, 1996). Belitsky & Sonenshein (1999) described an enhancer located 1.5 kb downstream of the 54 promoter of the rocG gene of the Bacillus subtilis. We are currently working on the construction of transcriptional fusions that should be helpful in a search for P6 activating conditions.
Since IHF binds in the rpoH regulatory region, it might be expected that it plays a role in rpoH transcription regulation. However, under the conditions of our experiment on in vitro transcription from P6, with PspF as an activator, IHF slightly inhibited the reaction (results not shown). Considering the results of Dworkin et al. (1998), who found that IHF facilitated transcription initiation only when a specific activator was used, it has to be accepted that, as long as the specific activator is unidentified, the significance of the IHF binding cannot be evaluated. Nevertheless, the IHF binding seems to be a strong argument for its regulatory role in transcription from P6. Nyström (1995) reported that IHF overproduction caused the induction of the rpoH-dependent heat shock response and proposed that this effect could reflect an influence of IHF on the activity of one or several rpoH promoters. Our results lend support to this notion.
One can speculate on the role of transcription from P6. Recently attention was paid to biofilm formation. This is an interesting process from the point of view of the development of unicellular organism cooperation leading to a kind of bacterial community. Beloin et al. (2004) found that biofilm formation involves, among other processes, the stress response. The pspA–E operon was strongly induced. The expression of ibpB and ibpA, genes for small heat shock proteins, increased 40- and 20-fold, respectively, compared with expression during the exponential growth phase of planktonic cells, in conditions inducing biofilm formation. The clpB and dnaK genes were also induced (Schembri et al., 2003). Elevated expression of these genes is dependent on 32, the rpoH gene product. The molecular mechanism of this induction remains obscure.
|
ACKNOWLEDGEMENTS |
|---|
grzyn for his critical reading of the manuscript. This work was supported by Grant 3 P04A 001 23 from the Polish Ministry of Scientific Research and Information Technology, State Committee for Scientific Research (to A. T.). Edited by: S. J. W. Busby
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Arsène, F., Tomoyasu, T. & Bukau, B. (2000). The heat shock response of Escherichia coli. Int J Food Microbiol 55, 3–9.[CrossRef][Medline]
Barrios, H., Valderrama, B. & Morett, E. (1999). Compilation and analysis of 54-dependent promoter sequences. Nucleic Acids Res 27, 4305–4313.
Belitsky, B. R. & Sonenshein, A. L. (1999). An enhancer element located downstream of the major glutamate dehydrogenase gene of Bacillus subtilis. Proc Natl Acad Sci U S A 96, 10290–10295.
Beloin, C., Valle, J., Latour-Lambert, P., Faure, P., Krzeminski, P., Balestrino, D., Haagensen, J. A., Molin, S., Prensier, G. & other authors (2004). Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression. Mol Microbiol 51, 659–674.[CrossRef][Medline]
Blattner, F. R., Plunkett, G., 3rd, Bloch, C. A., Perna, T. A., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K. & other authors (1997). The complete genome sequence of Escherichia coli K-12. Science 277, 1453–1474.
Brissette, J. L., Weiner, L., Ripmaster, T. L. & Model, P. (1991). Characterization and sequence of the Escherichia coli stress-induced psp operon. J Mol Biol 220, 35–48.[CrossRef][Medline]
Buck, M. & Cannon, W. (1992). Specific binding of the transcription factor sigma-54 to promoter DNA. Nature 358, 422–424.[CrossRef][Medline]
Cannon, W., Bordes, P., Wigneshweraraj, S. R. & Buck, M. (2003). Nucleotide-dependent triggering of RNA polymerase-DNA interactions by an AAA regulator of transcription. J Biol Chem 278, 19815–19825.
Carmona, M., Claverie-Martin, F. & Magasanik, B. (1997). DNA bending and the initiation of transcription at 54-dependent bacterial promoters. Proc Natl Acad Sci U S A 94, 9568–9572.
Connolly, L., Yura, T. & Gross, C. A. (1999). Autoregulation of the heat shock response in prokaryotes. In Molecular Chaperones and Folding Catalysts: Regulation, Cellular Function and Mechanisms, pp. 13–33. Edited by B. Bukau. Amsterdam: Harwood Academic Publishers.
Craig, N. L. & Nash, H. A. (1984). E. coli integration host factor binds to specific sites in DNA. Cell 39, 707–716.[CrossRef][Medline]
Darwin, A. J. (2005). The phage-shock-response. Mol Microbiol 57, 621–628.[CrossRef][Medline]
Dworkin, J., Jovanovic, G. & Model, P. (1997). Role of upstream activation sequences and integration host factor in transcriptional activation by the consitutively active prokaryotic enhancer-binding protein PspF. J Mol Biol 273, 377–388.[CrossRef][Medline]
Dworkin, J., Ninfa, A. J. & Model, P. (1998). A protein induced DNA bend increases the specificity of a prokaryotic enhancer-binding protein. Genes Dev 12, 894–900.
Elderkin, S., Jones, S., Schumacher, J., Studholme, D. & Buck, M. (2002). Mechanism of action of the Escherichia coli phage shock protein PspA in repression of the AAA family transcription factor PspF. J Mol Biol 320, 23–37.[CrossRef][Medline]
Elliott, T. & Geiduschek, E. P. (1984). Defining a bacteriophage T4 late promoter: absence of a "–35" region. Cell 36, 211–219.[CrossRef][Medline]
Erickson, J. W. & Gross, C. A. (1989). Identification of the E subunit of Escherichia coli RNA polymerase: a second alternative sigma involved in high temperature gene expression. Genes Dev 3, 1462–1471.
Erickson, J. W., Vaughn, V., Walter, W. A., Neidhardt, F. C. & Gross, C. A. (1987). Regulation of the promoters and transcripts rpoH, the Escherichia coli heat shock regulatory gene. Genes Dev 1, 419–432.
Filutowicz, M., Grimek, H. & Appelt, K. (1994). Purification of the Escherichia coli Integration Host Factor (IHF) in one chromatographic step. Gene 147, 149–150.[CrossRef][Medline]
Foster, J. E., Holmes, S. F. & Erie, D. A. (2001). Allosteric binding of nucleoside triphosphates to RNA polymerase regulates transcription elongation. Cell 106, 243–252.[CrossRef][Medline]
Fu, J., Gnatt, A. L., Bushnell, D. A., Jensen, G. J., Thompson, J. E., Burgess, R. R., David, P. R. & Kornberg, R. D. (2000). Yeast RNA-polymerase II at 5 Å resolution. Cell 98, 799–810.
Fujita, N. & Ishihama, A. (1996). Reconstitution of RNA polymerase. Methods Enzymol 273, 121–130.[Medline]
Goodrich, J. A., Schwartz, M. L. & McClure, W. R. (1990). Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res 18, 4993–5000.
Gralla, J. D. & Collado-Vides, J. (1996). Organization and function of transcription regulatory elements. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 1232–1246. Edited by F. C. Neidhart and others. Washington, DC: American Society for Microbiology.
Granston, A. E. & Nash, H. A. (1993). Characterization of a set of integration host factor mutants deficient for DNA binding. J Mol Biol 234, 45–49.[CrossRef][Medline]
Grimm, C., Aufsatz, W. & Panopoulos, N. J. (1995). The hrpRS locus of Pseudomonas syringae pv. phaseolicola constitutes a complex regulatory unit. Mol Microbiol 15, 155–165.[CrossRef][Medline]
Guo, Y., Wang, L. & Gralla, J. D. (1999). A fork junction DNA-protein switch that controls promoter melting by the bacterial enhancer-dependent sigma factor. EMBO J 18, 3736–3745.[CrossRef][Medline]
Guo, Y., Lew, C. M. & Gralla, J. D. (2000). Promoter opening by the 54 and 70 RNA polymerases: factor-directed alterations in the mechanism and tightness of control. Genes Dev 14, 2242–2255.
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[Medline]
Hankamer, B. D., Elderkin, S. L., Buck, M. & Nield, J. (2004). Organization of the AAA+ adaptor protein PspA is an oligomeric ring. J Biol Chem 279, 8862–8866.
Jishage, M., Kvint, K., Shingler, V. & Nyström, T. (2002). Regulation of factor competition by the alarmone ppGpp. Genes Dev 16, 1260–1270.
Jovanovic, G., Weiner, L. & Model, P. (1996). Identification, nucleotide sequence and characterization of PspF, the transcriptional activator of the Escherichia coli stress-induced psp operon. J Bacteriol 178, 1936–1945.
Jovanovic, G., Rakonjac, J. & Model, P. (1999). In vivo and in vitro activities of the Escherichia coli 54 transcription activator, PspF, and its DNA-binding mutant, PspFHTH. J Mol Biol 285, 469–483.[CrossRef][Medline]
Kallipolitis, B. H. & Valentin-Hansen, P. (1998). Transcription of rpoH, encoding the Escherichia coli heat-shock regulator 54, is negatively controlled by the cAMP-CRP/CytR nucleoprotein complex. Mol Microbiol 29, 1091–1099.[CrossRef][Medline]
Kuczyska-Winik, D., Laskowska, E. & Taylor, A. (2001). Transcription of the ibpB heat-shock gene is under control of 32 and 54-promoters; a third regulon of heat-shock response. Biochem Biophys Res Commun 284, 57–64.[CrossRef][Medline]
Laurie, A. D., Bernardo, L. M. D., Sze, C. C., Skärfstad, E., Szalewska-Paasz, A., Nyström, T. & Shingler, V. (2003). The role of the alarmone (p)ppGpp in N competition for core RNA polymerase. J Biol Chem 278, 1494–1503.
Leblanc, B. & Moss, T. (2001). DNase I footprinting. Methods Mol Biol 148, 31–38.[Medline]
MacNeil, D. (1981). General method, using Mu-Mudl dilysogens, to determine the direction of transcription and generate deletions in the glnA region of Escherichia coli. J Bacteriol 146, 260–268.
Maeda, H., Fujita, N. & Ishihama, A. (2000). Competition among seven Escherichia coli subunits: relative binding affinities to the core RNA polymerase. Nucleic Acids Res 28, 3497–3503.
Magnusson, L. U., Farewell, A. & Nyström, T. (2005). ppGpp: a global regulator in Escherichia coli. Trends Microbiol 13, 236–242.[CrossRef][Medline]
Merrick, M. J. (1993). In a class of its own – the RNA polymerase sigma factor 54. Mol Microbiol 10, 903–909.[Medline]
Messer, W. & Weigel, C. (1997). DnaA initiator – also a transcription factor. Mol Microbiol 24, 1–6.[CrossRef][Medline]
Missiakas, D. & Raina, S. (1998). The extracytoplasmic function sigma factors: role and regulation. Mol Microbiol 28, 1059–1066.[CrossRef][Medline]
Model, P., Jovanovic, G. & Dworkin, J. (1997). The Escherichia coli phage-shock-protein (psp) operon. Mol Microbiol 24, 255–261.[CrossRef][Medline]
Nagai, H., Yano, R., Erickson, J. R. & Yura, T. (1990). Transcriptional regulation of the heat shock regulatory gene rpoH in Escherichia coli: involvement of a novel catabolite-sensitive promoter. J Bacteriol 172, 2710–2271.
Nyström, T. (1995). Glucose starvation stimulon of Escherichia coli: role of integration host factor in starvation survival and growth phase-dependent protein synthesis. J Bacteriol 177, 5707–5710.
Nyström, T. (2004). Growth versus maintenance: a trade-off dictated by RNA polymerase availability and sigma factor competition. Mol Microbiol 54, 855–862.[CrossRef][Medline]
Pallen, M. (1999). RpoN-dependent transcription of rpoH? Mol Microbiol 31, 393.[CrossRef][Medline]
Pérez-Martin, J., Rojo, F. & de Lorenzo, V. (1994). Promoters responsive to DNA bending: a common theme in prokaryotic gene expression. Microbiol Rev 58, 268–290.
Polyakov, A., Severinova, E. & Darst, S. A. (1995). Three-dimensional structure of E. coli core RNA polymerase: promoter binding and elongation conformations of the enzyme. Cell 83, 365–373.[CrossRef][Medline]
Ramírez-Santos, J., Collado-Vides, J., Garcia-Varela, M. & Gómez-Eichelman, M. C. (2001). Conserved regulatory elements of the promoter sequence of the gene rpoH of enteric bacteria. Nucleic Acids Res 29, 380–386.
500 nt rpoH transcript. Activator proteins PspF (a), PspF