From the Institut für Allgemeine Mikrobiologie, Am Botanischen Garten 1-9, 24118 Kiel, Germany
Received for publication, September 10, 2002, and in revised form, October 9, 2002
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ABSTRACT |
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Archaea have a eukaryotic type of transcriptional
machinery containing homologues of the transcription factors
TATA-binding protein (TBP) and TFIIB (TFB) and a pol II type of RNA
polymerase, whereas transcriptional regulators identified in archaeal
genomes have bacterial counterparts. We describe here a novel regulator of heat shock response, Phr, from the hyperthermophilic archaeon Pyrococcus furiosus that is conserved among Euryarchaeota.
The protein specifically inhibited cell-free transcription of its own
gene and from promoters of a small heat shock protein, Hsp20, and of an
AAA+ ATPase. Inhibition of transcription was brought about
by abrogating RNA polymerase recruitment to the TBP/TFB promoter
complex. Phr bound to a 29-bp DNA sequence overlapping the
transcription start site. Three sequences conserved in the binding
sites of Phr, TTTA at All living organisms have developed molecular mechanisms to
protect themselves from the harmful effects of elevated temperatures and other stress factors. The overwhelming majority of information on
these heat-induced heat shock proteins
(Hsps)1 and heat shock genes
comes from bacteria and eukaryotes. In bacteria, several independent
mechanisms of regulation of heat shock promoters have been elucidated.
Regulation in Escherichia coli is brought about by the use
of alternative sigma factors that direct RNA polymerase to heat shock
promoters differing from the standard consensus promoter sequence (1).
A different mechanism operates in Gram-positive bacteria, which
involves binding and dissociation of a repressor to a DNA control
element upstream of the groEL and dnaK operons
(2). Recent analyses suggest that bacteria have evolved sophisticated
regulatory networks often combining positive and negative control
mechanisms to allow a time-tuned expression of heat shock genes (3). In
eukaryotes stress-induced transcription requires activation of a heat
shock factor, HSF, which binds as a trimer to the heat shock DNA
element, thereby stimulating transcription. The monomeric form of HSF
lacks both promoter binding and transcriptional activity (4).
Regulators of the heat shock response in the domain of Archaea have not
yet been identified, but studies of stress genes in archaeal genomes
revealed the presence of homologues of Hsp70/DnaK, Hsp60, Hsp40, GrpE,
and of small heat shock proteins (5). Hsp70 is only present in about
50% of the archaeal species inspected; GroEL and GroES seem not to
exist in Archaea (5). Their function in peptide folding is apparently
performed by the archaeal thermosome, which has been studied in some
molecular detail in Thermoplasma (6),
Methanopyrus (7), and Pyrodictium (8).
We are exploring the mechanism and regulation of gene transcription in
Pyrococcus furiosus growing optimally at 100 °C (9), using a cell-free transcription system (10). The transcriptional machinery of Archaea is eukaryotic-like. It consists of a pol II-type
of RNA polymerase, a TATA-box-binding protein (TBP), and a homologue of
the transcription factor TFIIB, TFB (11, 12).
Archaeal TBP and TFB interact with the archaeal promoter elements
TATA-box and B-recognition element (BRE) of an archaeal promoter in a
similar manner as their eukaryotic counterparts (13, 14). The
information on the regulation of transcription in Archaea is scarce but
in contrast to the eukaryotic nature of the basal archaeal
transcriptional machinery many homologues of bacterial regulators have
been identified in archaeal genomes (15, 16). A transcriptional
activator of gas vesicle proteins containing a leucine zipper motif has
been identified in the extreme halophilic archaeon Halobacterium
salinarum (17), although this protein did not show significant
sequence similarity to eukaryotic activators. Archaeal
homologues of the general bacterial regulator leucine responsive
regulatory protein, Lrp, have been shown to inhibit transcription from
their own promoters in vitro (18, 19). Since archaeal cells
do not have sigma factors or homologues of eukaryotic heat shock
factors (HSF) or sequences similar to heat shock factor elements (HSE)
the mechanism of control of heat shock response and the components
involved in this process are completely unclear.
We describe here an archaeal transcriptional repressor that shows, with
the exception of the region of its helix-turn-helix motif, no sequence
similarity to eukaryotic or bacterial regulators. It binds specifically
to the transcriptional start site of an archaeal heat shock gene
thereby inhibiting RNA polymerase recruitment. We also show that
mRNA levels of this regulator are induced after heat shock and
during stationary growth phase. By contrast, levels of the regulator
protein were only slightly elevated during heat stress.
Cultivation of Cells and Preparation of Total RNA--
P.
furiosus cells were grown at 95 °C in a 100-liter fermentor in
marine medium as described previously (9). To study the effects of heat
shock on the transcription of the hsp20, the
aaa+ atpase, and the putative heat
shock regulator (phr) genes, the temperature during
logarithmic growth phase (OD578 of 0.5) was shifted to
103 °C. Before heat shock treatment and after 30, 60, and 90 min at
103 °C, aliquots of 2 liters of culture broth were taken and rapidly
cooled down using a heat exchange device. Cells were harvested by
centrifugation at 5000 rpm for 10 min at 4 °C. The sediment was
resuspended in TBS (300 mM NaCl, 20 mM
Tris-HCl, pH 7.6) and stored at
To analyze the effects of growth phase on the expression of heat shock
genes, Pyrococcus cells were grown at 95 °C in a
100-liter fermentor, and aliquots of 6.2, 4.5, 3.0, and 2.0 liters were taken at an OD578 nm of 0.14, 0.27, 0.6, and 0.58, respectively. Cells were harvested as described above. Total RNA was
isolated from cells grown at 95 °C at different stages of the
"growth curve" and from heat shocked cells following the protocol
of DiRuggiero and Robb (20).
Cloning and Expression of Phr--
The gene encoding
phr was amplified by PCR using oligonucleotide primers
pftrpexp1-F (5'-GGAATTCAATATGGGAGAGGAGCTAAACAG-3') and pftrpexp1-R
(5'-CGCGGATCCTTAAATGGTAATGTTTAGG-3'), which include the restriction
sites for BamHI and NdeI, respectively. The
resulting 625-bp PCR fragment and the plasmid pET19b (Novagen) were
hydrolyzed with BamHI and NdeI. The fragment was
cloned into pET19b, and the sequence of the insert was verified by DNA
sequencing. For expression of the recombinant histidine-tagged Phr, the
plasmid was transformed into E. coli strain BL21CP(DE3)-RIL
(Stratagene). Cells were grown in LB medium with 200 µg/ml ampicillin
and 68 µg/ml chloramphenicol at 37 °C while shaking. When an
OD600 nm of 0.6 was reached, expression was induced by the
addition of isopropyl-1-thio-
For gel shift analyses with oligonucleotides, Phr that did not contain
a His tag was used. Expression of the protein was refined as described
above. The protein was overexpressed in E. coli BL 21-CP(DE3)-RIL carrying plasmid pET17b containing the phr
gene sequence as insert and a pLysE plasmid. Cells were lysed in 50 mM sodium phosphate buffer (pH 7.2) using a French pressure
cell at 140 MPa. The cell extract was incubated at 70 °C for 15 min and after centrifugation at 40,000 rpm (Beckman L60 ultracentrifuge, rotor 70 Ti) additionally at 75 °C for 15 min. After a further centrifugation step, the supernatant was applied to a heparin affinity
column (HiTrap Heparin, Amersham Biosciences) and eluted using a salt
gradient ranging from 0 to 1000 mM NaCl. Fractions containing Phr were pooled and concentrated by ultrafiltration using a
YM3 Membrane (Millipore). 2 ml were applied to a gel filtration column
(Superdex 200 16/60, Amersham Biosciences). Gel filtration was carried
out in 50 mM sodium phosphate buffer containing 150 mM NaCl (pH 7.2). Fractions containing Phr were
concentrated by cation exchange chromatography using a sulfopropyl
Sepharose column (SP XL, Amersham Biosciences). Bound proteins were
eluted by a linear salt gradient ranging from 0 to 1000 mM
NaCl. Phr was eluted at an approximate salt concentration of 500 mM NaCl.
To determine the native molecular mass of Phr the protein was applied
to a Superdex 200 16/60 column. The column was calibrated using
apoferritin (443 kDa), Plasmid Construction--
The P. furiosus
aaa+ atpase, hsp20, and
phr promoters were amplified by PCR using primers ATPF1
(5'-GTTGAATAGCTTTAGTACCC-3') and ATPR1 (5'-CTCAACAACATCTCCTGGTG-3') for
the aaa+ atpase promoter, 20F3
(5'-CTTCGTGGAGAGAATTTACTG-3') and 20R1 (5'-CTTGGCCTGCTGAAGAATTC-3') for
the hsp20 promoter and PfPr1F1 (5'-GCTGGAGTTACCGTTGTTC-3')
and PfPr1R (5'-GCTCGCTGACAAAGTAAGG-3') for the phr promoter.
The PCR products were ligated into SmaI-digested pUC18
(pUC19 in the case of cloning the phr promoter). This
resulted in the plasmid constructs upatpase,
uphsp20, and upphr. The sequences of the archaeal
DNA in the plasmids were confirmed by DNA sequencing. Plasmid DNA was
purified by centrifugation in CsCl density gradients as described
previously (22).
In Vitro Transcription--
In vitro transcription
assays were performed as described previously (10). Assays were
composed of 300 ng of linearized template DNA (XbaI-digested
uphsp20 or upphr or KpnI-digested upatpase), 0.012 µM Pyrococcus RNA
polymerase (RNAP), 0.076 µM recombinant TFB, 0.68 µM recombinant TBP, and different amounts (0.37-1.5
µM) recombinant Phr in a 25-µl reaction mixture. The control template was XbaI-digested plasmid pUC19 containing
the P. furiosus gdh promoter sequence from Primer Extension--
For analyses of the transcriptional start
sites in vitro, cell-free transcription reactions with
unlabeled precursors (0.44 mM each) were performed. The
end-labeled primers used for the reactions were: ATPR1
(5'-CTCAACAACATCTCCTGGTG-3'), complementary to nucleotides +139 to +159
of the aaa+ atpase gene, 20R1
(5'-CTTGGCCTGCTGAAGAATTC-3'), complementary to nucleotides +116 to +136
of the hsp20 gene and PfPr1R (5'-GCTCGCTGACAAAGTAAGG-3'), complementary to nucleotides +107 to +126 of the phr gene.
Primer extension reactions were performed as described previously (10). For in vivo RNA analyses primer extension was performed with
30 µg of total RNA of P. furiosus. The RNA was isolated
from cells cultured under different growth conditions as described
above. Primer extension products were quantified using a PhosphorImager.
Preparation of Probes for EMSA--
DNA probes for EMSA were
generated by PCR using the end-labeled primers ATPF2
(5'-GGTTCTATTATCAATTAATTCC-3') and M13/pUCrev (5'-GAGCGGATAACAATTTCACACAGG-3'), and plasmid upatpase as
template. The PCR products were excised from a 6% native
polyacrylamide gel, eluted in TE buffer (10 mM Tris-HCl, pH
7.0, 01.mM EDTA) and precipitated with ethanol.
Oligonucleotides (MWG Oligo) were hybridized as follows: 3000 pmol of
complementary oligonucleotides (sequence of the noncoding strand shown
in Fig. 6B) dissolved in hybridization buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.2 M NaCl) were heated to 95 °C and allowed to cool down to
room temperature overnight. Double-stranded DNA was purified via native
gel electrophoresis on a 15% polyacrylamide gel. Double-stranded DNA
was excised from the gel, the gel slices were immersed in gel elution
buffer (1 mM Tris-HCl, pH 7, 0.5 M
NH4Ac, pH 7) and eluted overnight at 37 °C. Eluted DNA
was precipitated with ethanol and resuspended in TE buffer, pH 8. Double-stranded DNA fragments were radiolabeled using T4 polynucleotide
kinase (MBI Fermentas) according to the MBI protocol. DNA was purified by extraction with phenol/chloroform/isoamyl alcohol (25:24:1) and
chloroform/isoamyl alcohol (24:1) and precipitated with ethanol.
EMSA--
The 10-µl binding reaction mixture contained 40 mM HEPES-NaOH, pH 7.3, 325 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 5%
polyethylene glycol (PEG) 8000, 3 µg of bovine serum albumin, and 1 µg of poly(d(IC)) as nonspecific competitor DNA.
Each reaction contained 1-10 ng of labeled DNA. Binding reactions were
incubated at 70 °C for 30 min and contained 0.23 µM recombinant TBP, 0.13 µM recombinant TFB, 0.008 µM RNAP, and 0.74, 1.9, or 3.7 µM
recombinant Phr. DNA protein complexes were analyzed on nondenaturing
5% acrylamide gels buffered in Tris borate/EDTA buffer (TBE) (23).
DNase I Footprinting--
Probes containing the promoter region
of the aaa+atpase gene were generated
by PCR using a combination of one unlabeled primer and a second primer
that was end-labeled with T4 polynucleotide kinase and
[ Preparation of Antibodies Against Recombinant Pyrococcus
Phr--
Purified native His-tagged Phr (500 µg) was used for
immunization of a rabbit using the standard protocol (Eurogentec,
Seraing, Belgium). The preimmune and anti-Phr IgG fractions were
isolated from the serum by affinity chromatography on protein
A-Sepharose (Amersham Biosciences).
Western Blot Analysis--
0.2 g (wet weight) of P. furiosus cells harvested before and after heat shock were
suspended in PBSEG buffer (50 mM sodium phosphate, pH 7, 150 mM NaCl, 5 mM EDTA, 10% glycerol) and
lysed by sonication (Branson Sonifier 250). Cell debris was pelleted by
centrifugation, and 20 µg of the cell-free extract were separated by
electrophoresis on a 12% SDS-polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (Immobilon P, Millipore) by semi-dry blotting and detected
immunologically using purified anti-Phr IgG essentially as described
previously (24). The relative amount of Phr was quantified using a PhosphorImager.
Phr Is a Specific Regulator of Archaeal Heat Shock Genes--
A
putative heat shock regulator from P. furiosus, Phr, is
composed of 202 amino acids encoded by an open reading frame of 609 nucleotides (GenBankTM accession no. Q8U030). It has a
predicted pI of 7.68, a molecular mass of 24,034, and forms a dimer in
solution (see "Experimental Procedures"). The His-tagged protein
showed an apparent molecular mass of 26,000 under denaturating
conditions (Fig. 1). Comparison of the
amino acid sequence of Phr with putative heat shock regulators from
other Archaea revealed that the related Pyrococcus strains P. abyssi and P. horikoshii have the highest
percent identity and similarity to Phr (Table
I). The putative regulator of a moderate
thermophilic methanogenic archaeon Methanothermobacter thermoautotrophicus shows lower percent identity and of the
hyperthermophilic archaeal sulfate reducer Archaeoglobus the
lowest percent identity. The availability of a cell-free transcription
system for P. furiosus allowed us to investigate the
function of Phr as transcriptional regulator.
To characterize these putative archaeal heat shock genes, the promoter
regions of the Pyrococcus aaa+ atpase
and hsp20 gene and of the Phr were cloned by PCR and used as
templates in cell-free transcription reactions. The in vitro transcription start sites were identified by primer extension analyses
of RNA products synthesized by the cell-free Pyrococcus system. All these templates showed typical archaeal TATA-boxes and
BRE-elements (Fig. 2B). As
expected, they were expressed in the cell-free system (Fig.
2A). Transcription initiated at a distance of 24, 23, and 20 nucleotides downstream of the TATA-box at a G, T, or A residue,
respectively.
To study the effect of Phr on the expression of these genes the protein
was added to cell-free transcription reactions containing different
putative heat shock genes as templates, and the labeled RNA products
were analyzed directly in denaturing gels. When increasing amounts of
Phr were added to the templates encoding hsp20, the aaa+ atpase or to the gene encoding
the regulator Phr itself, synthesis of the run-off transcripts was
reduced at lower concentrations and strongly or completely inhibited at
higher concentrations of Phr (Fig. 3,
A and C). The inhibitory activity of Phr was
highest at the aaa+ atpase promoter
(complete inactivation at a molar ratio DNA: regulator is 1:125;
addition of 500 ng) and lowest at the phr promoter (no
complete inhibition at a molar ratio of 1:250; addition of 1000 ng).
The effect of Phr on cell-free transcription of the P. furiosus glutamate dehydrogenase promoter (Fig. 3B) was
studied as a control. Addition of Phr did not affect the rates of
transcription from this promoter. These findings suggest that Phr is a
negative regulator of heat shock response.
Phr Inhibits Transcription by Abrogating RNA Polymerase
Recruitment--
To investigate the interaction of Phr with the
archaeal transcriptional machinery in more detail Phr was added to DNA
binding reactions containing the aaa+
atpase promoter, TBP/TFB, and RNAP. Electrophoretic mobility shift analyses showed that Phr bound to a labeled DNA fragment encoding
the DNA region from position
To analyze the DNA binding region of Phr and of Pyrococcus
transcriptional components at the
aaa+atpase promoter in molecular
detail, DNase I footprinting experiments were performed. Phr protected
the DNA region from Identification of Cis-acting Sequences Required for Phr
Binding--
To identify the structural determinants of archaeal heat
shock promoters required for operator recognition by Phr, binding experiments with short double-stranded oligonucleotides containing the
wild-type aaa+ atpase DNA binding
region and mutated sequences were performed. Inspection of sequences in
the Phr binding site, which are conserved between the three promoters
investigated revealed the existence of three common signals: The
consensus sequences TTTA at
To analyze the DNA sequences recognized by Phr in more detail we
carried out a mutational analysis of binding motifs within the 36-bp
fragment. Mutation of three nucleotides in a nonconserved DNA region
located at position Transcription of the aaa+ atpase and phr Is Induced
under Heat Shock Conditions and Stimulated during Stationary Growth
Phase--
The in vitro data provided in this study
demonstrate inhibition of transcription of putative archaeal heat shock
genes by an archaeal regulatory protein and suggest that Phr is a
negative regulator of heat shock response in Pyrococcus. To
provide additional experimental evidence that the investigated genes
are real heat shock genes and that the function of the regulator is
related with stress, the expression of the aaa+
atpase and of the phr gene was
investigated under various physiological conditions. P. furiosus cultures were grown in a 100-liter fermentor at 95 °C,
cells were heated to 103 °C for 30, 60, and 90 min, and the mRNA
levels were analyzed by primer extension experiments. In a separate set
of experiments the mRNA levels of the same genes were analyzed
during different growth phases.
Analysis of aaa+ atpase mRNA
showed that the RNA levels in Pyrococcus cells were
increased after 30 min of heat shock treatment by a factor of 25 (Fig.
7A). After 60 and 90 min of
heat shock treatment, the mRNA levels were still elevated by a
factor of 11 and 13, compared with cells grown at 95 °C. Also the
mRNA levels of the phr gene were dramatically induced by
treatment of cells at 103 °C. After 30 min, the mRNA level was
42 times higher than in nonstressed cells. The phr mRNA
levels were even more increased after prolonged heat shock treatment by
a factor of 49 after 60 min and a factor of 50 after 90 min (Fig.
7A).
The transcript levels of both genes were also increased during the
stationary growth phase. Compared with early and late exponential growth phase, expression of the aaa+
atpase gene was stimulated by a factor of 1.5 and 3 during
early and late stationary phase, that of the phr gene by a
factor of 6 and 18 (Fig. 7A). Control experiments using the
P. furiosus gdh gene showed no increase of mRNA levels
after heat shock and during stationary growth phase (data not shown).
Taken together, these data provide evidence that the
aaa+ atpase gene is activated by heat
shock and starvation, and that Phr is a regulator of heat shock and
potentially also of general stress response.
Analysis of the protein levels of Phr by Western blotting showed that
the expression of the regulator was only slightly enhanced after heat
shock and remained constant during different growth phases (Fig.
7B). Thus, in contrast to Hsp20 from P. furiosus that was only synthesized in significant amounts in heat-shocked cells
(26), Phr is both expressed during growth at the optimal temperature
and after temperature upshift. This finding indicates that Phr is
required in nonstressed and stressed cells.
Several lines of evidence indicate that we have identified an
archaeal regulator of heat shock response. A protein conserved among
Euryarchaeota (Ref. 27 and Table I) was found to inhibit specifically
cell-free transcription of the aaa+
atpase gene, of the gene encoding the small archaeal heat
shock protein Hsp20, and of its own gene (Fig. 3). The protein bound specifically to the promoter region of the aaa+
atpase promoter (Figs. 4 and 5). The data shown in this
study indicated that this specific DNA binding site overlapped with the
RNAP binding site (Fig. 5) and that promoter-bound Phr inhibited RNAP
recruitment at heat shock promoters (Fig. 4). Within the DNA binding
site of Phr three DNA signals conserved among the promoters
investigated here were identified (Fig. 2B). These three sequences were shown to be essential for binding of Phr to DNA by
mutational studies (Fig. 6). Finally, the aaa+
atpase, the phr gene, and the small heat shock
gene hsp20 (Ref. 26; see below) produced highly elevated
mRNA levels after heat shock treatment (Fig. 7). These results
demonstrated that the genes regulated in vitro by Phr are
clearly affected by heat shock treatment in vivo.
The cellular function of the P. furiosus AAA+
ATPase is not known but members of the AAA+ superfamily are
often involved in energy-dependent proteolysis, molecular
chaperone-like activities (28), and transcriptional activation (29).
The small heat shock proteins (sHsps) from Archaea have been studied to
some extent. The sHsp from Methanococcus jannaschii is a
16.5-kDa protein that forms a unique spherical oligomer of 24 subunits
and has the ability to protect proteins in E. coli extracts
from thermal aggregation (30, 31). Hsp20 from P. furiosus
(that is identical with Pfu-sHSP described by Laksanalamai et
al., Ref. 26) the product of the gene investigated in this
study, showed a sequence similarity of 33.8% with M. jannaschii sHsp and of 43 and 31% with two sHsps of bacterial
origin (26). Both P. furiosus Hsp20 and its mRNA were
shown to be induced by heat shock. More than 90% of cellular proteins
from E. coli remained soluble after 40 min at 105 °C when
Hsp20 from P. furiosus was overexpressed. In addition, Hsp20
prevented a mesophilic glutamate dehydrogenase from aggregation as a
result of heat treatment in vitro. These results (26)
demonstrate clearly that the product of the P. furiosus
hsp20 gene investigated in our study has chaperone activity.
mRNA levels of the aaa+ atpase
and of the gene phr were shown in this work to be elevated during heat shock. These findings support the conclusion that the
DNA-binding protein and DNA signals described in this work can be
considered as trans- and cis-acting elements of archaeal heat shock reponse.
Most heat inducible archaeal genes seem to contain the typical archaeal
promoter sequences consisting of a TATA-box at The data presented here suggest that the regulators and
regulatory sequences of heat shock control differ between Archaea, bacteria, and eukaryotes. Phr is a negative regulator that binds to an
operator overlapping the transcription start site characterized by
three different and separated DNA sequences (Fig. 2B).
Transcription of class I heat shock genes in Gram-positive bacteria is
also regulated by a repressor. These genes show a completely different cis-acting regulatory sequence consisting of a 9-bp inverted repeat separated by a 9-bp spacer, frequently located between the
transcriptional and translational start sites (controlling
inverted repeat of chaperone
expression, CIRCE; Ref. 2). The eukaryotic heat shock factor, HSF, is a positive regulator binding to repeating arrays of the
5-bp HSE sequence nGAAn. The number of HSE elements can vary, but each
repeat is inverted with respect to the immediately adjacent repeat and
the repeats are located upstream of the TATA-box between positions Also during the stationary growth phase the levels of the
aaa+ atpase and of phr
mRNA were significantly increased (Fig. 7A) compared
with logarithmic phase suggesting that the transcription of these genes
is also affected by starvation and general stress. Since the promoter
of the aaa+ atpase gene contains a
Phr binding site and binding of Phr prevents RNA polymerase recruitment
at this promoter (Fig. 4) it is likely that Phr is also involved in the
growth phase-dependent regulation of this gene. Protein
levels of the AAA+ ATPase were not analyzed here. The
finding that Phr protein levels remained constant during various growth
phases (Fig. 7B) indicates again that an additional hitherto
unknown mechanism modulating its binding activity seems to be required
for regulation of stress response in Archaea. The identification of the
transcriptional regulator described here offers the opportunity to
address exciting questions concerning the crystal structure of Phr and
its interplay with cellular factors and proteins. These studies will be
important not only for studies of the mechanism of heat shock
regulation in Archaea but will also contribute a deeper
understanding of the evolution of stress response in all three
domains of life.
10, TGGTAA at the transcription start site, and
AAAA at position +10, were required for Phr binding and are proposed as
consensus regulatory sequences of Pyrococcus heat shock
promoters. Shifting the growth temperature from 95 to 103 °C caused
a dramatic increase of mRNA levels for the
aaa+ atpase and phr
genes, but expression of the Phr protein was only weakly stimulated.
Our findings suggest that heat shock response in Archaea is
negatively regulated by a mechanism involving binding of Phr to
conserved sequences.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
-D-galactopyranoside to a
final concentration of 0.4 mM. After incubation for 3 h at 30 °C, cells were harvested by centrifugation and resuspended
in PING buffer (50 mM sodium phosphate, 10 mM
imidazole, 300 mM NaCl, 10% glycerol, pH 8.0). Cells were
then disrupted by a French pressure cell (American Instruments) at 140 MPa, and the cell-free extract was centrifuged at 40,000 rpm for 1 h. The supernatant was loaded on a Ni-nitrilotriacetic acid-agarose
column (Qiagen), and the column was washed with 10 volumes of PING
buffer containing 20 mM imidazole and 1 M NaCl. The recombinant protein was eluted by a linear increasing imidazole gradient (20 mM to 0.5 M imidazole in PING
buffer containing 300 mM NaCl) and eluted from the column
at 300 mM imidazole. Fractions of 0.5-ml size were
collected and tested for the presence of Phr by SDS-PAGE. Protein
concentration was determined by the method of Bradford (21).
-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic anydrase (29 kDa) as standards. Phr showed an
apparent molecular mass of 48,000 during gel filtration chromatography
in 50 mM phosphate buffer. This finding indicated that
recombinant Phr forms a dimer in solution.
95 to +163
(10). The reactions were assembled at 4 °C and then incubated at
70 °C for 30 min. The transcripts were analyzed by denaturing
polyacrylamide gel electrophoresis as described previously (22).
Transcriptional activities were quantified using a PhosphorImager (FLA
5000 Fuji, Aida Imaging Software, Raytest).
-32P]ATP. For the nontemplate strand labeled ATPF2
(5'-GGTTCTATTATCAATTAATTCC-3') and unlabeled M13/pUCrev
(5'-GAGCGGATAACAATTTCACACAGG-3') and for the template strand labeled
ATPR1 (5'-CTCAACAACATCTCCTGGTG-3') and unlabeled M13/pUCf
(5'-GCCAGGGTTTTCCCAGTCACGA-3') were used. The probe for footprinting
analysis of the nontemplate strand contained the DNA region from
95
to +225, the second probe the DNA sequence from
236 to +160. For
purification, PCR products were excised from a native 6%
polyacrylamide gel and eluted in TE buffer, pH 7 overnight at room
temperature. After ethanol precipitation, about 10 ng of this DNA was
used per footprinting reaction in a total volume of 10 µl. Buffer
conditions were identical to those used for EMSA, except that in the
footprinting reactions CaCl2 was added to a final
concentration of 0.1 mM. DNaseI footprinting reactions
contained 0.93 µM TBP, 0.55 µM TFB, and/or
3.7 µM Phr. Binding reactions were preincubated at
70 °C for 30 min and then 100 milliunits of DNaseI were added. After
30 s of DNaseI treatment at 70 °C, the reaction was stopped by
the addition of 18 µl of a solution containing 1.5 M
NH4Ac, 70 mM EDTA, pH 8.0 and 114 ng/µl tRNA.
The samples were extracted with phenol and precipitated with ethanol.
The DNA fragments were resuspended in formamide-loading buffer (98%
formamide, 0.1% bromphenol blue, 0.1% xylene cyanol, 10 mM EDTA, pH 8.0) and were electrophoresed on a 6%
denaturing polyacrylamide gel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of the archaeal heat shock
regulator, Phr. 12% SDS-PAGE of purified N-terminal
histidine-tagged Phr (lane 2) stained with Coomassie Blue.
Molecular weight standards are shown in lane 1.
Amino acid similarity of Phr from P. furiosus to archaeal homologues
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Fig. 2.
Analyses of the transcription start sites at
P. furiosus heat shock promoters and at the promoter
of the heat shock regulator. A, primer extension
reactions were performed using the products of in vitro
transcription assays. The position of the primer extension products are
indicated by arrows. A, T, G, and C lanes correspond to the
dideoxynucleotide sequencing reactions carried out using the same
oligonucleotide primers as in primer extension reactions. The sequences
of the template strands are shown on the right side of the
figures with the transcription start sites shown in bold
letters. B, summary of the primer extension data,
showing the sequences of the aaa+
atpase, hsp20, and phr promoters. The
transcription start sites (+1) are shown in bold with
arrows. The positions of the TATA elements and BRE are
boxed. Sequence similarities among the three promoters are
underlined.
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Fig. 3.
Phr inhibits cell-free transcription of
archaeal heat shock genes and of its own promoter. Linearized
plasmid DNA containing the aaa+
atpase, hsp20, and gdh promoter
regions of P. furiosus were used as templates in cell-free
transcription experiments. The run-off transcripts are indicated by
arrows. The reactions contained 0, 250, 500, or 1000 ng of
Phr as indicated on the top of the lanes, which correspond
to 0, 0.37, 0.75, and 1.5 µM, respectively. Cell-free
transcription assays performed with the aaa+
atpase and hsp20 promoters (A), the
gdh promoter (B), and the phr promoter
(C) as templates. Transcriptional activities were quantified
using a PhosphorImager. Transcription activity in the absence of Phr
equals 100%.
95 to +225 (Fig.
4, lanes 1 and 2).
No shift was observed when a DNA fragment containing the gdh
promoter region was used (data not shown), indicating that binding of
Phr to the aaa+ atpase promoter was
specific. TBP/TFB formed a complex with lower electrophoretic mobility
(Fig. 4, lane 3). Addition of RNA polymerase (RNAP) to
binding reactions containing TBP/TFB resulted in the formation of a
third, slowly migrating complex (lane 5). We have recently
shown the presence of TBP/TFB and of RNAP in similarly migrating
complexes formed at the lrpA promoter of P. furiosus by serological analyses (19). We therefore assume that
these components are also present in the corresponding complexes
observed at the aaa+ atpase promoter.
When Phr was added to binding reactions containing TBP/TFB, binding of
the archaeal transcription factors was not affected, but a third
complex of lower mobility was observed (lane 4). This
finding and the decrease in intensity of the band corresponding to the
Phr-DNA complex in lane 4 suggest that this third complex consisted of
TBP/TFB and Phr. When Phr was added in low concentrations (molar ratio
RNAP:Phr of 1:92) to binding reactions containing both TBP/TFB and
RNAP, the Phr-DNA, the TBP/TFB-DNA, the TBP/TFB-Phr-DNA, and the
TBP/TFB-RNAP-DNA complexes were observed (lane 6). However, addition of increasing amounts of Phr to the binding reaction containing TBP/TFB and RNAP decreased the amount of the
TBP-TFB-RNAP-DNA complex and increased the amount of the
TBP/TFB-Phr-DNA complex (molar ratio RNAP:Phr of 1:237 in lane
7 and 1:462 in lane 8). This finding supports the
conclusion that binding of Phr to DNA did not affect binding of
TBP/TFB, but inhibited association of RNAP with the TBP/TFB promoter
complex.
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Fig. 4.
Phr specifically binds at the
aaa+ atpase promoter and
prevents RNAP recruitment. EMSA was performed with the
radiolabeled aaa+ atpase promoter DNA
using 0.23 µM TBP, 0.13 µM TFB, 0.008 µM RNAP, and 0.74 (+), 1.9 (++), or 3.7 µM
(+++) Phr, as indicated. Free DNA and protein-DNA complexes are
indicated to the left.
15 to +14 on the nontemplate (Fig.
5A, left panel,
lanes 1-4) and from
17 to +12 on the template DNA strand
(Fig. 5A, right panel, lanes 1-4) from DNase I digestion.
The TBP/TFB footprint extended from position
41 to
17 on the
nontemplate (Fig. 5A, left panel, lanes 5 and
6) and from
36 to
19 (Fig. 5A, right
panel, lanes 5 and 6) on the template DNA
strand. The TBP/TFB-binding site was centered at the TATA-box and Phr
bound to the DNA region overlapping the transcription start site (Fig.
5B). This DNA segment downstream of the TBP/TFB binding site
has been shown to be bound by the archaeal RNAP (25). This finding
suggests that Phr and TBP/TFB can bind simultaneously to the same DNA
molecule. To investigate this both Phr and TBP/TFB were added to
binding reactions. Phr and TBP/TFB induced a footprint both on the
nontemplate and template DNA strand (Fig. 5A,
left and right panel, lanes 7 and
8). The extension of the protected DNA regions was the same
as with individual components. On the nontemplate DNA strand a
hypersensitivity site located at position
6 was observed in complexes
containing both TPB/TFB and Phr (left panel, lanes
7 and 8). Therefore, binding of Phr to the region of
the transcription start site prevents the association of RNAP with the
TBP/TFB promoter complex.
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Fig. 5.
Phr binding site at an archaeal heat shock
promoter. A, DNaseI footprinting analyses of TBP/TFB
and of Phr at the nontemplate strand (left panel) and
template strand (right panel) of the
aaa+ atpase promoter. DNaseI
footprints were performed using 200 ng (0.93 µM) of TBP,
200 ng (0.55 µM) of TFB, or 1000 ng (3.7 µM) of Phr, as indicated. DNA fragments were analyzed in
parallel with a sequencing reaction by denaturing gel electrophoresis.
The position of the TBP/TFB- and Phr-induced footprints relative to the
start site of transcription are indicated on the right side
of the figure. The asterisk marks the hypersensitivity site
at position 6. B, summarized footprinting data, showing
the sequence of the aaa+ atpase
promoter. The TATA box and the transcription start site (+1) are shown
in bold letters. Gray rectangles indicate the
TBP/TFB induced DNaseI footprints, and white rectangles
indicate the Phr-induced footprints.
10, TGGTAA at the transcription start
site, and AAAA centered at position +10 (Fig. 2B). DNA
fragments of 36 and 30 bp containing these three conserved elements
were sufficient for Phr binding (Fig. 6A, lanes 3 and
4 and 7 and 8). Phr did not form a
complex with single-stranded 36-nucleotide DNA (Fig.
6A, lanes 5 and 6) indicating that
double-stranded DNA is required for association of Phr with promoter
DNA. A 25-bp fragment containing the conserved sequences was not bound
by Phr (Fig. 6A, lanes 1 and 2). As no conserved sequences were deleted in this fragment we assume that not the removed
5'- and 3'-flanking DNA sequences were essential, but that a minimal
length of DNA between 26 and 30 nucleotides is required for specific
binding of Phr.
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Fig. 6.
Identification of Phr binding signals at an
archaeal heat shock promoter. A, binding of Phr to
different sized DNA fragments. The DNA fragments represent the Phr
binding site of the aaa+ atpase gene
promoter. The three conserved sequences of heat shock promoters are
shown in bold letters in the lower part of the
figure. BRE and the TATA box are boxed, the vertical
arrows indicate the boundaries of the Phr footprint. Binding
reactions were performed with radiolabeled DNA for 30 min at 70 °C;
complexes were analyzed by native gel electrophoresis (EMSA).
B, binding of Phr to DNA fragments car- rying certain mutations. Mutations and nucleotides, which are
conserved among the binding sites of the aaa+
aptase, the hsp20, and the phr gene
are indicated below the figure. The three conserved putative
binding signals are shown in bold letters in the wild type
sequence. Mutated bases are underlined. The percentage of
the various templates bound by Phr is indicated on top of
the figure.
17 to
20 decreased the binding ability of Phr
only slightly from 66 to 56% (Fig. 6B, lanes 2 and
3, compare wild type and control). Mutation of three
nucleotides of the
10 motif (mutV), of three nucleotides of the
initiator site motif (mutM), and three nucleotides of the AAAA motif
(mutH) reduced the binding activity of Phr to 2.5, 9, and 29% (Fig.
6B, lanes 4-6), respectively. This result identified the
10 region as most important for operator recognition by Phr and
therefore, the effects of single point mutations in this DNA segment
were analyzed. Each of the T residues of the TTTA motif was replaced by
a C, the A residue by a G (Fig. 6B, mut1-mut 4). Analysis
of the binding ability of these transition mutants revealed that each
nucleotide of this position contributed significantly to DNA binding
(Fig. 6B). The first and third T residues were more important for complex formation than the second T residue and the A
residue. These studies provide biological evidence for the importance
of the three conserved binding motifs in the regulatory region of Phr
and identify the TTTA motif at
10 as crucial for Phr binding.
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Fig. 7.
Regulation of mRNA and Phr synthesis in
vivo. A, transcription of aaa+
atpase and phr is strongly induced under heat
shock conditions and stimulated during stationary growth phase in
vivo. Primer extension analyses of 30 µg of P. furiosus RNA isolated from cells cultured under different growth
conditions. Lane 1, culture growing at 95 °C; lanes
2, 3, and 4, cultures after heat shock
treatment at 103 °C for 30, 60, and 90 min., respectively.
Lanes 5-8, nonstressed cultures with OD578 of
0.14, 0.27, 0.60, and 0.58, which correspond to the early exponential,
late exponential, early stationary, and late stationary phases,
respectively. Left panel, analysis of the
aaa+ atpase transcripts; right
panel, analysis of the phr transcripts. The relative
intensities of primer extension signals were quantified using a
PhosporImager. The strongest signal equals 100%. B, Phr
protein levels increased after heat shock and remained constant during
different growth phases. Phr was analyzed in a 20-µg cell extract of
P. furiosus by Western blotting. Left panel,
different growth temperatures; lane 1, 20 µg of
recombinant Phr; lane 2, culture growing at 95 °C;
lanes 3, 4, and 5, cultures after heat
shock treatment at 103 °C for 30, 60, and 90 min, respectively.
Right panel, different growth phases; lane 1, 20 µg of recombinant Phr; lanes 2-5, nonstressed cultures
harvested at OD578 of 0.14, 0.27, and 0.58, respectively.
The relative amount of Phr was quantified using a PhosphorImager and is
indicated on the top of the figure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
25 and a purine-rich
BRE element immediately upstream of the TATA-box (5). The cis-acting
sequences specific of archaeal heat shock promoters are poorly defined.
The regulatory sequence elements of genes encoding heat inducible
proteins of the chaperonin-containing TcP-1 family from the halophilic
Archaeon Haloferax volcanii have been identified. The
analysis of this extreme halophile revealed that heat induced
transcription in vivo required sequences upstream and
downstream of the TATA-box (32). A specific consensus binding site for
a regulator was not inferred by these authors, but similar to our study
the regulatory signals were located in the core promoter region. The
three regulatory DNA sequences identified in this study are conserved
within the putative Phr DNA binding region of at least two heat shock
genes of P. furiosus and in the promoter region of the
regulator itself (Fig. 2B) whose transcription also appears
to be dramatically induced by heat shock (Fig. 7A). As Phr
is conserved among methanogenic Archaea and Archaeoglobus (Table I) it is likely that also similar recognition sequences exist on
DNA level of these organisms. We propose here the sequences TTTA at
10, TGGTAA at the transcription start site, and AAAA at +10 as
consensus sequence for P. furiosus heat shock promoters. A
similar sequence was also found in the promoter region of the thermosome-encoding gene of P. furiosus (data not shown).
40
and
270 (33). Considering the extensive similarities of the basal
transcriptional machineries of Archaea and eukaryotes these distinct
differences are initially surprising. However, the few characterized
archaeal regulators like Lrp and the metal-dependent
regulator MDR1 from A. fulgidus (34) resemble in
sequence and mechanism of action bacterial regulators. The lack of
sequence similarity of the archaeal regulator Phr
with bacterial and eukaryotic regulators and the absence of homologues of sigma factors and eukaryotic HSF in Archaea pose the intriguing possibility that Archaea have evolved a unique mechanism to control the
heat shock response. This mechanism is unclear but this work suggests
that binding of Phr to heat shock promoters during normal growth and
dissociation of the repressor after heat shock is crucial for
transcriptional regulation of the hsp20 and
aaa+ atpase genes. Phr is denatured
at 103 °C in
vitro,2 and dissociation
of the denatured protein from its operator sequence may account for the
high increase of phr mRNA detected after temperature upshift (Fig. 7). Our finding that heat shock triggered the
accumulation of Phr mRNA dramatically but that of the regulator
protein only slightly (Fig. 7, A and B) suggests
that regulation of archaeal heat shock response is complex, and that
more than one regulator might be involved. Regulation on the level of
mRNA stability and/or translation might occur, and a protein or
cofactor modulating the binding activity of Phr might exist.
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ACKNOWLEDGEMENTS |
---|
We thank Jens Thomsen for the initial help in cloning of Phr from genomic DNA of P. furiosus, and the valuable advice of Winfried Hausner in cloning and bioinformatic work is appreciated. We thank Uta Wehmeyer for technical assistance and Holger Preidel for large scale cultivation of P. furiosus.
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FOOTNOTES |
---|
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft priority program and from the Fonds der Chemischen Industrie (to M. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Universität
Regensburg, Universitätsstr. 31, 93040 Regensburg, Germany. Tel.: 49-941-943-3160; Fax: 49-941-943-2403; E-mail: mthomm@ifam.uni-kiel.de or Michael.Thomm{at}Biologie.Uni-Regensburg.de.
Published, JBC Papers in Press, October 14, 2002, DOI 10.1074/jbc.M209250200
2 M. Thomm, A. Engelmann, G. Vierke, and R. Ladenstein, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: Hsps, heat shock proteins; pol II, polymerase II; EMSA, electrophoretic mobility shift assay; TBP, TATA-box-binding protein; BRE, B-recognition element; RNAP, Pyrococcus RNA polymerase; TFB, transcription factor IIB (TFIIB); Phr, Pyrococcus heat shock regulator.
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REFERENCES |
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