kle. 1*:t}W ; Na42,.200811., pp.17-20 Research Reports of the School of Engineering,
K nki University No42, 2008, pp.17.20
Amino acid sequence diversities extremely halophilic archaeon
in TBP, TATA binding protein, of Haloarcula japonica strain TR-1
Hironari MATSUMI and Kaoru NAKASONE
Synapsis
The TATA-box binding protein (TBP) is a basal transcription factor involved in transcription initiation in Eukarya and Archaea. Through exhaustive analyses of the whole geneme of extremely halophilic archaeon, Haloar•cula japonica strain TR-1, six TBP genes were found and structurally analyzed. These TBPs were designated as TBP1, TBP2, TBP3, TBP4, TBP5 and TBP6, respectively and these TBPs were diverged from other archaeal TBPs that have been known. TBP1 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 35.0 % identity to that of H nrarism rtui. TBP2 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 36.5 % identity to that of H marismortur: TBP3 gene was found to encode a polypeptide consisting of 186 amino acid residues, showing 100 % identity to that of H. marismortui. TBP4 gene was found to encode a polypeptide consisting of 185 amino acid residues, showing 42.9 % identity to that of H. rnarismartui. TBP5 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 35.4 % identity to that of H marisnrortw TBP6 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 46.1 % identity to that of H marisinortui. By phylogenetic analyses of these six proteins, TBP3 is conserved in amino acid sequence with other archaeal strains including methanogens and thermophiles, It may suggest that the TBP3 is core TBP function in transcriptional initiation such as housekeeping genes. Six histidine-tagged version of the H japonica TBPs were produced in Escherichia coli in a denature conditions after construction of overexpression plasmids and purified by means of Ni-chelating chromatography.
Key word: Haioarrula japonica, TATA-box binding protein, TBP, overexpression
I. Introduction
Until recently, a long-established dogma in biology was that all life on earth is conventionally divided into two kingdoms: eukaryotes and prokaryotes.
However, 16S rDNA analyses in the 1970s indicated that the prokaryotic world is not a single coherent entity, but, instead, is divided into two distinct groups, the eubacteria and archaea 1) Whereas a large amount of research has been directed towards determining the mechanism and regulation of transcription in Eukarya and Eubacteria, relatively little work has been conducted on archaeal transcription systems.
Over the past decades, however, several lines of evidence have suggested that the transcription systems of archaea and eucarya are fundamentally homologous, including RNA polymerase and basal transcription factors of TBP and TFB.
Halophilic archaea survive in environments containing molar concentrations of salts.
The high external salt condition is balanced by an equally high internal salt concentration. Thus, halophilic archaea offer the opportunity to study specific interactions of biomolecules (i.e. DNA and proteins) in the presence of high osmolarity. Thus, as a prerequisite for studying haloarchaeal transcription initiation, we report here the cloning six tbp genes form H. japonica and heterologous overexpression and purification of these TBPs.
2. Materials and Methods 2.1 Strain and culture conditions
The H japonica strains used in this study are listed hi Table 1. The IL japonica cells were grown at 37°C with vigorous shaking in a medium, containing (per litter): 200 g of NaC1, 20 g of Meal. 4H20, 3.0 g of trisodium citrate • 2H2O, 3.0 g of KC1, 10 g of yeast extract, 7.5 g of casamino
Graduate School of Systems Engineering, Kink' University
17
acid and a mixture of trace elements (0.0218 g of MnC12.4H2D and 0.486 g of FeC13.6H2O per 100 m1). E. coli JM199 was the host for all experiments and was incubated at 37°C in LB broth (Table 1).
2.2 DNA manipulations
H japonica genomic DNA was isolated with Microbial DNA isolation kit (MoBio) as recommended by the manufacture. The plasmids used in this study are listed in Table 1. The plasmids used in this study are listed in Table 1, and the sequences of the PCR primers used are given in Table 2. Plasmids were subcloned and maintained in Escherichia call using JM109 (TaKaRa).
2.3 Construction of expression plasmids and overexpression studies
The coding region of TBP genes from H. japonica TR-1, was PCR amplified using the genomic DNA
as template (Table 3). The oligonucleotides were complementary to the 5'- and 3'- ends of the genes
and contained the restriction sites BamHI at the 5'.
end and HindIII at the 3'- end. The PCR products
were cloned into the pT7Blue2 T-vector (TaKaRa)
and transformed into E. coilJM109 competent cell (TaKaRa). Purified plasmids from the recombinants identified through blue-white selection, were digested with BamHI and HindIII, and cloned into the corresponding restriction sites of the expression vector pCold I (TaKaRa). For expression of hexahistidine-tagged fusion proteins (TBP1, 2, 3, 4, 5 and 6), the plasmids were transformed into JM109, The cells were grown at 37°C in Terrific Broth. At mid-log phase, the cultures were shifted to 15°C and IPTG (1 mM) was added to induce protein expression.
Overexpressed products were purified by Ni-NTA affinity chromatography, and they were checked by SDS-PAGE,
2.4 Homology modeling of the TBP
Homology modeling of the TBP was carried out using RasMol software
(http://www.umass.edulmicrobiolrasmoll).
Amino acid sequence diversities in TBP, TATA binding protein. of extremely halophilic archacon Naaarcida japonica strain TR-1 19
3. Results and discussion
At first, exhaustive analyses of the whole genome of extremely halophilic archaeon, Haloarcula japonica strain TR-1 were carried out to search genes for TATA-box binding protein (TBP) for a basal transcription factor involved in transcription initiation. The analyses revealed six TBP genes and these were found to encode in the several replicons of the strain TR-1. These TBPs were then designated as TBP1, TBP2, TBP3, TBP4, TBP5 and TBP6, respectively. Structural analyses of these proteins showed that these TBPs were diverged from other archaeal TBPs have been already known (Fig. 1). TBP1 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 35.0 % identity to that of H.
inarismortui TBP2 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 36.5 % identity to that of H.
inarisrnortui. TBP3 gene was found to encode a polypeptide consisting of 186 amino acid residues, showing 100 % identity to that of H.
rrrarismortui. TBP4 gene was found to encode a polypeptide consisting of 185 amino acid residues, showing 42.9 % identity to that of H.
marisrrrortur: TBP5 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 35.4 % identity to that of H.
inarisinor tui T13P6 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 46.1 % identity to that of H inarisrnartui By phylogenetic analyses of these six proteins, TBP3 is conserved in amino acid sequence with other archaeal strains including methanogens and thennophiles (Fig. 1). It may suggest that the TBP3 is core TBP function in transcriptional initiation such as housekeeping genes and also TBP3 is common factor in archaeal domain.
Fig. 1 Phylogenetic tree of TBPs in archaea Fig. 2 Genetic organization of TBP3 gene with other archaeal strains
Red names show halophilic archaea, green show methanogenic archaea and blue show therinophilic archaea. Phylogenetic tree was constructed based on amino acid sequence, and tree was drawn using the neighbor-joining algorism.
20 AA1:. NQ,t2
r N CI 65
a. a.a.
yC{amal
2 _ + - + - ia.
H a.
F- [fl a
. I H
(kDa) 85-- 60 - 50 — 40 — 25 — 20 — 15—
+ - + 4
Fig. 3 Homology modeling of H. japonica TBP3 Fig. 4 Overexpression in R coil and purification of H. japonica TBPs.
Marker; Pre-stained Protein Marker (Nacalai Tesque),
—; The cell lysates containing His -tag protein, -F;
His-tag purification product The cell lysates containing His-tag protein and his-tag purification
product were separated on 15% SDS-PAGE. GeI were stained with Cooinassie Brilliant Blue.
To compare genetic organization of TBP3 gene of the strain TR-1 with other archaeal strains, alignments of several sequences containing TBP3 similar genes were performed. The genetic organization of tbp3 gene was not conserved in many archaeal strains expect for H. ma#isinortul (Fig. 2). Homology modeling of the TBP was also carried out using RasMol software to visualize acidic region of the protein. Figure 3 shows the visualization of TBP3 protein and color in red shows acidic residues of the T13P3, as expected in halophilic archaeon. These comparison of model visualization with other TBPs is required for the further analyses to know structural and functional importance in these six TBPs.
In order to facilitate TBP purification after heterologous overproduction in E. coil, six tbp genes were coned into the expression vector pCold I, resulting in the fusion of hexahistidine tagged to the 5-end and the N-terminal addition of peptides to these Tl3Ps may be unlikely to interfere with its function, Induction of expression in a transgenic.~' coil strain led to a massive production of the fusion proteins, hexahisticline-tagged TBPs (Fig. 4). It was possible to purify these fusion proteins in a one step purification with nickel-chelating sepharose
column (Fig. 4). These recombinant proteins will be useful to reconstitute in vitro transcription system under high salt conditions, with haloarchaeal RNA polytnerase.
4. References
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Oshima, Pyrimidine biosynthesis genes (f)yrIe and pyr•l']of an extreme thermophile, Tbeiinus therrrrophilus, Appl. Environ. Microbial ., 62 (6), 2191.2194 (1996)
2) T. Takashina, K. Gtozaki, T. Hamamoto , and K.
Horikoshi, Isolation of halophilic and halotolerant bacteria from a Japanese salt field and comparison of the partial 16S rRNA gene sequence of an extremely halophilic isolate with those of other extreme haIophiles, Biodiv. Conserv., 3, 632-642 (1994)
3) J. Soppa, and T. A. Link, The TATA-box-binding protein (TBP) of Jialobacterium salinarinn Cloning of the tbp gene, heterologous production of TBP and folding of TBP into a native conformation, Eur. J . Biochemn., 249, 318-324 (1997)
