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

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 sulinurum 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 70) 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-boxcontaining DNA fragment, indicating that the fusion protein can be used to characterize DNA recognition by the halobacterial TBP.Keywords: Archaea ; Halobacterium salinarurn ; transcription factor; TATA-box-binding protein ; TATAbox.The mechanisms of promoter recognition and transcription initiation are different in Bacteria and Eucarya. In Bacteria, promoter recognition is accomplished by a RNA polymerase containing a tightly bound 0 factor. In Eucarya, promoter recognition is mediated by a variety of transcription factors, which direct RNA polymerase to the promoter. The TATA-box binding protein (TBP) is the most general eucaryal transcription factor, it is essential for transcription initiation with all three RNA polymerases (Kim and Roeder, 1994). The binding of TFIID, a complex of TBP and TBP associated factors (TAF), to the TATA-box of RNA polymerase I1 promoters is the first step in transcription initiation. TFIIB is the next factor to enter the preinitiation complex, before additional factors and polymerase I1 are recruited.Recently, it became evident that TBP and TFIIB are not confined to the domain Eucarya, but that homologous proteins exist Correspondence to J. Soppa, J. W.

Regulation of the expression of gas vesicle genes in Haloferax mediterranei: interaction of the two regulatory proteins GvpD and GvpE

“…The two Gvp‐his proteins were synthesized in E. coli and purified under denaturing conditions. The Gvp‐his proteins were refolded by dialysis against buffers containing decreasing urea, but increasing KCl concentrations up to 2.5 M. A similar and successful refolding has been demonstrated with the TATA‐box binding protein TBP of H. salinarum (Soppa and Link, 1997), and also with the glucose dehydrogenase of H. mediterranei , where the enzymatic activity was rapidly recovered from buffers with 8 M urea by 40‐fold dilution and addition of 2–3 M KCl or NaCl (Pire et al ., 2001).…”

From genomes to function: haloarchaea as model organisms

“…A fourth possibility is to use an alternative method, if techniques are intrinsically salt sensitive. Examples are the use of co-affinity isolation instead of co-immunoprecipitation (Zimmermann & Pfeifer, 2003) or a pull-down approach instead of an electrophorectic mobility shift assay to analyse DNA protein interactions (Soppa & Link, 1997).…”

Expression of multiple tfb genes in different Halobacterium salinarum strains and interaction of TFB with transcriptional activator GvpE

“…All TFB His proteins and GvpH His were successfully puriWed under denaturing conditions (8 M urea) by Ni-NTA aYnity chromatography. The proteins were dialyzed against decreasing urea and raising KCl concentrations to obtain the native, salt-adapted form (Soppa and Link 1997). TfbA His and TfbE His precipitated during this procedure and could not be kept in solution under any of the conditions tested.…”