Binding of four repressors to double-stranded tet operator region stabilizes it against thermal denaturation

Abstract

The investigation of protein–DNA interactions benefits from methods for the dissection of chromosomal DNA into handy fragments and their subsequent preparation in large amounts^1,2. Of particular interest are proteins active in gene regulation and their interaction with the control sequences of the respective genes³. Recently, we reported⁴ the purification of the molecular components of the control elements from the tetracycline-resistance (tet) gene located on the transposon Tn10. The Tet repressor inhibits transcription of the tet gene and its own gene⁴. When the tet operator was prepared on a 187 base pair (bp) DNA fragment⁴, the Tet repressor was found to bind specifically to this fragment with a stoichiometry of four Tet repressors per DNA fragment⁴. Tetracycline inhibits this binding⁴ and operates in vivo as an inducer for the expression of the Tn10-encoded tetracycline resistance^5,6. We now report thermal denaturation experiments of the Tet repressor–tet operator complex and demonstrate independently that four Tet repressor molecules bind to the 187 bp DNA and stabilize a 125 bp double-stranded DNA sequence against thermal denaturation.