The TATA-Box-Binding Protein (TBP) of Halobacterium Salinarum

Cloning of the tbp Gene, Heterologous Production of TBP and Folding of TBP into a Native Conformation

Authors


  • Note. The nucleotide sequence data published here have been sub-mitted to the EMBL sequence data bank and are available under accession number X93206.

J. Soppa, J. W. Goethe-Universität, Biozentrum Niederursel, Institut für Mikrobiologie, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany
Fax:+ 49 69 798 29 564.
E-mail:Soppa@em.uni-frankfurt.de

Abstract

The TATA-box binding protein (TBP) is a basal transcription factor involved in transcription initiation in Eucarya and Archaea. Using a tbp-specific probe, a 4.5-kbp genomic fragment from Halobacterium salinarum was cloned and sequenced. It contained the tbp gene and the 5’-ends of two additional open reading frames, but surprisingly, 70% of the cloned fragment (3.2 kbp) was devoid of coding capacity or similarity to database sequences.

The deduced halobacterial TBP exhibits sequence similarities to other archaeal (41–43%) as well as to eucaryal (27–38%) TBP. A comparative analysis showed that the archaeal and eucaryal TBP form two related monophylic protein families, and the archaeal TBP possess features which separate them from eucaryal TBP. Compared with the other TBP, the halobacterial TBP is unique in having a high excess of negatively charged residues.

A histidine-tagged version of the halobacterial TBP was produced in Escherichia coli in a denatured conformation and purified by means of Ni-chelating chromatography. CD spectroscopy was used to monitor TBP secondary structure and the conditions necessary for folding it into a native conformation. In the absence of denaturating agents, the folded as well as the unfolded state were found to be stable over a wide range of salt concentrations. Properly folded TBP was shown to bind to a halobacterial TATA-box-containing DNA fragment, indicating that the fusion protein can be used to characterize DNA recognition by the halobacterial TBP.

Abbreviations.
TBP

TATA-box-binding protein

TAP

TBP-associated factor

NCS

nickel-chelating Sepharose

Ancillary