©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Translation Product of the Presumptive Thermococcus celer TATA-binding Protein Sequence Is a Transcription Factor Related in Structure and Function to Methanococcus Transcription Factor B (*)

Winfried Hausner Michael Thomm (§)

From the Institut fr Allgemeine Mikrobiologie, Christian-Albrechts-Universitt zu Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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()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.


EXPERIMENTAL PROCEDURES

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 pKS30416 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).


RESULTS

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 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.


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 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.



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 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 pKS30416) 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.


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 pKS30416 (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.



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 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.




DISCUSSION

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.()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.

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) .


FOOTNOTES

*
This work was supported by the European Community Biotechnology Program, a grant of the Deutsche Forschungsgemeinschaft, and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-431-880-4334; Fax: 49-431-880-2194; Nam01{at}rz.uni-kiel.d400.de

The abbreviations used are: TBP, TATA-binding protein; aTFB, archaeal transcription factor B; aTFA, archaeal transcription factor A.

H. P. Gohl, B. Grndahl, and M. Thomm, manuscript in preparation.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.