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.
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.
Soppa
J
J
Max-Planck-Institut für Biochemie, Martinsried, Germany. Soppa@em.uni-frankfurt.de
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D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells; D019766:Genes, Archaeal; D019617:Halobacterium salinarum / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D016385:TATA Box; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D019843:Archaeal Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors