From the
A gene for a putative homolog of TATA-binding protein (TBP) from Thermococcus celer has been expressed in Escherichia
coli, and the function of the purified recombinant protein was
studied in a Methanococcus-derived cell-free transcription
system. Thermococcus TBP can replace archaeal transcription
factor B (aTFB) in cell-free transcription reactions. This
transcriptional activation is TATA box-dependent and occurs both on
tRNA The genomes of hyperthermophilic Archaea Pyrococcus woesei and Thermococcus celer include sequences that would
encode proteins similar to eucaryal transcription factor IIB (1, 2) and TBP
To address the question whether the putative homolog of TBP
in Thermococcus genome shows transcription factor activity in vitro, this gene was overexpressed in E. coli, and
the function of the recombinant polypeptide was investigated in a Methanococcus cell-free system. In Methanococcus, two
transcription factors, aTFA and aTFB, and RNA polymerase are required
for specific cell-free transcription from a tRNA
Figure 1:
Thermococcus TBP directs accurate
cell-free transcription by Methanococcus RNA polymerase. The
presence and absence of the individual components of the Methanococcus cell-free transcription system and of Thermococcus TBP (TcTBP) in transcription reactions
are indicated on top of the lanes by a + and - sign,
respectively. Transcription reactions contained Methanococcus transcriptional components as described under ``Experimental
Procedures'' and 200 ng (lane10) or 400 ng (lane11) of Thermococcus TBP. RNA products
transcribed from a supercoiled plasmid (pIC31/2) (9) harboring
the Methanococcus tRNA
To investigate whether transcription activation mediated
by Thermococcus TBP is brought about by the TATA box, template
activities of several archaeal promoters harboring a TATA box were
assayed together with promoter mutants of a tRNA
Figure 2:
TATA box-dependent activation of
transcription by Thermococcus TBP from Methanococcus and Methanothermus promoters. aTFB was substituted by Thermococcus TBP (TcTBP) in transcription reactions
as indicated at the top of the lanes. The
run-off transcripts from linearized pIC31/2, pIC31/44, pIC31/61, and
pKS304
Thermococcus TBP produced in E. coli showed an
apparent molecular mass of 23.5 kDa (Fig. 3A) when
analyzed by denaturing gel electrophoresis. A polyclonal antibody
raised against this polypeptide was probed with protein immunoblots of
the three transcriptional components of the Methanococcus cell-free system. Both aTFA fraction and RNA polymerase showed no
serological cross-reaction (Fig. 3B, lanes 1 and 2). However, in the aTFB fraction, a single band
corresponding to a molecular mass of 28 kDa bound antibodies (Fig. 3B, lane3). The molecular mass
of this cross-reacting polypeptide is identical to the molecular mass
of aTFB determined by SDS-polyacrylamide gel
electrophoresis(10) . This finding demonstrates that Thermococcus TBP and aTFB are structurally related.
Figure 3:
Thermococcus TBP and Methanococcus aTFB are structurally related. A, analysis of purified Thermococcus TBP (TcTBP). A purified Thermococcus TBP Superdex 200 fraction (600 ng) was electrophoresed on a 12%
SDS-polyacrylamide gel followed by staining with Coomassie Blue. B, immunological cross-reaction of Thermococcus TBP
and aTFB. Western blots of Methanococcus transcriptional
components aTFA (5 µl, lane1), RNA polymerase (aPol, 2.5 units; lane2), aTFB (0.75 units, lane3), and Thermococcus TBP (TcTBP, 6 ng, lane4) were challenged with antibodies
raised against Thermococcus TBP. Binding of antibodies was
visualized by peroxidase-coupled antibodies. The gel was calibrated by
using a prestained protein standard
(Bio-Rad).
To
provide further evidence for a functional similarity between Thermococcus TBP and aTFB, the effect of the antiserum
directed against Thermococcus TBP on cell-free transcription
by the Methanococcus system was studied. When increasing
amounts of purified IgG were added in preincubation reactions to aTFB
fraction, specific transcription was inhibited, which depended on the
amount of antibodies added to the reactions (Fig. 4, lanes
2-5). Preincubation with IgG purified from preimmune serum
led to a weak, presumably nonspecific inhibition of transcription, in
particular at high concentrations (Fig. 4, lanes
6-9; compare with control in lane1).
However, this inhibition was clearly less than that observed in the
presence of specific antibodies (Fig. 4, lanes6-9, compare with lanes 2-5).
This finding unequivocally demonstrates that the activity in Methanococcus aTFB fraction and that of Thermococcus TBP are identical.
Figure 4:
Inhibition of Methanococcus cell-free transcription by anti-Thermococcus TBP
antibodies. In vitro transcription reactions were
reconstituted from Methanococcus transcriptional components
aTFB, aTFA, and RNA polymerase and assayed for synthesis of
pre-tRNA
We describe here the interaction of recombinant Thermococcus TBP with Methanococcus transcriptional
machinery. The findings presented provide evidence that the gene that
exhibits sequence similarity to eucaryal TBP, which was detected
recently in the genome of Thermococcus, encodes an archaeal
transcription factor that has a function similar to that of aTFB.
First, Thermococcus TBP can substitute for aTFB in a Methanococcus-derived cell-free system (Fig. 1).
Second, activation of transcription brought about by this polypeptide
requires a TATA box (Fig. 2, lane4). TATA
boxes are essential elements of archaeal
promoters(7, 13) , and recently the TATA box has been
identified as the binding site for aTFB. The translation products of open reading frames
encoding putative TBPs from hyperthermophilic Archaea (3, 4) give a molecular mass of 21,000 daltons and do
not show the N-terminal extension encountered in eucaryal
TBPs(14) . The molecular mass of Methanococcus aTFB
determined by electrophoresis under denaturing conditions was 28,000
daltons(10) , and this polypeptide showed serological
cross-reaction with anti-Thermococcus TBP antibodies (Fig. 3B). Hence, aTFB most likely contains the
N-terminal extension missing in TBPs from hyperthermophiles. The
serological cross-reaction between Methanococcus aTFB and Thermococcus TBP indicates a close relationship of these
polypeptides also at the structural level. The demonstration of
similar function and structure of a Thermococcus and Methanococcus transcription factor provided here clearly
indicates homology of these polypeptides. We therefore suggest that one
designates aTFB as Methanococcus TBP. Our finding that Thermococcus TBP shows an activity directing specific
transcription from archaeal TATA boxes strongly supports the conclusion
that the genomes of Archaea encode transcription factors closely
related to eucaryal TBPs and provides biochemical evidence for the
hypothesis that archaeal and eucaryal transcriptional machineries are
basically homologous(3, 4, 5) .
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and protein-encoding genes as templates indicating
that Thermococcus TBP is a general transcription factor.
Antibodies raised against Thermococcus TBP bind to Methanococcus aTFB and inhibit aTFB activity. These findings
demonstrate that Thermococcus TBP (like eucaryal TBPs) can
direct specific transcription from TATA boxes.
(
)of
transcription factor IID(3, 4) . Genes of archaeal
TBPs encode two repeated domains that show 42-38% identity to the
sequence repeats of human TBP, respectively(4) . The protein
translation of the gene encoding Pyrococcus TBP was shown to
bind to the TATA box of a putative homologous promoter and seems also
to be expressed in Pyrococcus cells(3) , suggesting
homology of eucaryal and archaeal TBPs. However, an actual function of
the gene products of TBPs from hyperthermophiles in transcription has
not been demonstrated as a Pyrococcus transcription system has
not yet been described. We have shown recently that yeast and human TBP
can functionally replace archaeal transcription factor B in a Methanococcus thermolithotrophicus cell-free transcription
system(5) . M. thermolithotrophicus is a moderate
thermophile that can grow still at temperatures close to the lower
temperature range of growth of Thermococcus(6) .
Hence, a cell-free system derived from this organism may prove useful
to investigate also the function of transcription factors from
hyperthermophiles. We show here that recombinant Thermococcus TBP substitutes for Methanococcus aTFB in transcription
reactions and provide immunochemical evidence for a structural
relationship between these proteins. Our data imply a similar function
for aTFB, Thermococcus TBP, and eucaryal TBPs.
Templates for Cell-free Transcription
Reactions
The standard template used was plasmid pIC31/2
containing the tRNA gene of Methanococcus
vannielii. pIC31/44 is a single-point mutant of this template
containing a T
G transversion at position -26. pIC31/61
contains a substitution of internal sequences of tRNA
from position 5 to 88 by Escherichia coli DNA(5, 7) . Plasmid pKS304
16 harbors the
promoter region of the gene encoding the histone hmf1 from Methanothermus fervidus that shows a tandem repeat of the TATA
box(8) . Linearized templates for analyses of run-off
transcripts were prepared as described previously(5) . Plasmid
DNA was purified by repeated centrifugation in CsCl density gradients
as described previously(9) .
Purification of Transcription Factors and RNA
Polymerase
RNA polymerase and aTFB were purified as described
previously(10) . aTFA was isolated by phosphocellulose
chromatography (9) . Residual processing activity was
eliminated by heating the aTFA fraction for 15 min at 95 °C and
subsequent removal of precipitated material by centrifugation. aTFA
activity is heat-stable and remains in the supernatant.Expression and Purification of Recombinant Thermococcus
TBP
A DNA fragment encoding the gene of Thermococcus TBP was amplified by polymerase chain reaction using the
oligonucleotides 5`-CGTCTTAC(CATATG)AGCAATGTAAAGCTCAG-3` and
5`-GTCATTC(GAATTC)ACCACTCCTCTTCTTCCCC-3`. The DNA fragment was
hydrolyzed with NdeI and EcoRI (restriction sites of
the oligonucleotides are in parentheses) and ligated between the NdeI and EcoRI sites of the expression vector pET14b
(Novagen, Madison, WI). The resulting plasmid pTBPTc.14 contained the
coding region of the TBP from Thermococcus and a His-tag at
the N terminus of the protein under the control of an inducible T7
promoter(11) . The construct was transformed into E. coli BL21(DE3). The expression of the protein was induced by the
addition of isopropyl-1-thio--D-galactopyranoside (final
concentration, 1 mM) to a growing culture (OD = 0.7).
The recombinant protein was purified using affinity chromatography on
immobilized Ni
columns according to the instructions
of Novagen. Thermococcus TBP fractions were pooled for further
purification, diluted with 3 volumes of TN
buffer (50
mM Tris/HCl, 50 mM NaCl, 10% glycerol, pH 8.0), and
applied to a Mono Q column (HR 5/5, Pharmacia Biotech Inc.). Proteins
were eluted with a linear gradient (15 ml/50-1000 mM NaCl). Fractions that were able to replace aTFB activity in a Methanococcus cell-free transcription system were pooled and
further purified by chromatography on a Superdex 200 column (HR 16/60,
Pharmacia) equilibrated with TN
buffer (50 mM Tris/HCl, 300 mM NaCl, 10% glycerol, pH 8.0).
In Vitro Transcription Assays
100-µl
transcription reactions were performed as described
previously(9) . Reactions contained 10 µl of the
phosphocellulose fraction of aTFA, 5 µl of Superdex fraction of RNA
polymerase (5 units; (10) ), and 10 µl of Superdex fraction
of aTFB (1.5 units; (10) ). In experiments investigating the
effect of anti-Thermococcus TBP IgG on transcription, aTFB was
preincubated in a total volume of 50 µl with various quantities of
anti-Thermococcus TBP or preimmune IgG fractions for 30 min at
room temperature, and the reaction was completed by the addition of
aTFA, RNA polymerase, and ribonucleoside triphosphates.Preparation of Antibodies against Recombinant
Thermococcus TBP
Purified protein (500 µg) was used for
immunization of a rabbit. Immunization was performed by Eurogentec
(Seraing, Belgium) following the standard immunization protocol. The
anti-Thermococcus TBP and the preimmune IgG fractions (each
2.5 mg/ml) were isolated from the serum by affinity chromatography on
protein A-Sepharose (Pharmacia).Western Blot Analysis
Various protein fractions of Methanococcus were separated by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose membranes (BA-S 85,
Schleicher & Schuell) by semi-dry blotting. The membrane was
incubated for at least 1 h or overnight in TBST buffer (50 mM Tris/HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) containing
5% (w/v) nonfat dried milk (Gluecksklee, Nestle Frankfurt). After
blocking, the membrane was incubated with either preimmune or
anti-Thermococcus TBP IgG for 1 h. IgG was diluted 1:333 in
TBST buffer. Unbound antibodies were removed by washing three times
with TBST buffer for 10 min. After this step, the membrane was
incubated with a 1:2000 dilution of peroxidase-labeled anti-rabbit IgG
(from sheep) for 1 h. The membrane was washed then with TBST buffer
three times for 15 min. Chemiluminescence detection was performed
according to the instructions of the manufacturer (Boehringer
Mannheim).
promoter (7, 12) (Fig. 1, lanes 1-8).
When aTFA was replaced by Thermococcus TBP, the system was
incapable of synthesizing a specific RNA product (Fig. 1, lane9). However, substitution of aTFB by Thermococcus TBP resulted in synthesis of pre-tRNA
(Fig. 1, lane10). Increasing the amount
of Thermococcus TBP in the transcription reactions led to
increased synthesis of specific RNA products (Fig. 1, lanes
10 and 11). This finding demonstrates that Thermococcus TBP shows transcription factor activity and
suggests homology of Methanococcus aTFB and Thermococcus TBP.
gene were analyzed by
denaturing polyacrylamide gels and autoradiography. The arrow to the right marks the pre-tRNA
of M. vannielii. aPol, RNA polymerase; b, bases.
gene in
the Methanococcus/Thermococcus hybrid transcription
system. Analysis of run-off transcripts from a template containing a
promoter-down single-point mutation of the TATA box (pIC31/44; Fig. 2) revealed that the latter was essential for aTFB (7) (Fig. 2, lane3) and Thermococcus TBP (Fig. 2, lane4)
mediated initiation of transcription. As expected, deletion of internal
sequences of tRNA
(pIC31/61) did not affect expression by
the Methanococcus cell-free system (7) (Fig. 2, lane5) and the Methanococcus/Thermococcus hybrid system (Fig. 2, lane6). RNA products were also
synthesized by both systems with high activity from the promoter of
histone hmf1 (plasmid pKS304
16) from M. fervidus containing two tandem TATA boxes (Fig. 2, lanes 7 and 8) and from the promoter of nifH1 gene from M. thermolithotrophicus encoding an archaeal nitrogenase (data
not shown). These findings indicate that Thermococcus TBP
recognizes promoters of archaeal protein-encoding genes.
16 (see ``Experimental Procedures'') were analyzed
as described in Fig. 1. The sizes of run-off transcripts from
linearized pIC31/2 and pIC31/61 are 89 and 47 nucleotides from the
tandem promoters of the hmf1 gene 155 and 81 nucleotides,
respectively. aPol, RNA polymerase; b,
bases.
from plasmid pIC31/2. Anti-Thermococcus TBP (anti-TcTBP) IgG and preimmune IgG were preincubated
with aTFB fraction. The reactions contained 0, 2.5, 5, 10, and 20
µg of anti-Thermococcus TBP IgG (lanes 1-5)
or 2.5, 5, 10, and 20 µg of preimmune IgG (lanes
6-9). Transcriptional analysis was performed as described in
the legend of Fig. 1.
(
)Third, TATA boxes of protein-encoding archaeal genes
are recognized by Thermococcus TBP (Fig. 2, lane8) indicating that it acts (like aTFB(10) ) as a
general transcription factor. Finally, antibodies raised against Thermococcus TBP bind to aTFB in Western blots (Fig. 3B) and inhibit aTFB-dependent transcription in
the Methanococcus cell-free transcription system (Fig. 4). Our finding that the functions of aTFB and Thermococcus TBP are very similar indicates a high degree of
conservation of the protein between these two Euryarchaeota(15) although the genera Methanococcus and Thermococcus show considerable phylogenetic divergence (16) and the two species M. thermolithotrophicus and Thermococcus celer a different optimum growth
temperature(6, 17) . The observation that a Thermococcus transcription factor is able to direct specific
transcription by Methanococcus RNA polymerase is in line with
our recent finding that yeast and human TBP can substitute for aTFB in
the Methanococcus cell-free system(5) . Hence, Methanococcus transcriptional machinery appears extremely
conserved and highly suitable to detect similarities of transcriptional
components originating from evolutionary unrelated organisms at the
functional level.
We would like to thank Dr. Roger Garrett (Institute
of Molecular Biology, Copenhagen) for comments on the manuscript, Drs.
Terrance Marsh, Claudia Reich, and Gary Olsen (Department of
Microbiology, University of Illinois at Urbana Champaign) for providing
the gene encoding Thermococcus TBP before publication, Dr.
Carl Woese (Department of Microbiology, University at Urbana Champaign)
for stimulating discussions, and Jutta Kock for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.