Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region

Abstract

The promoter and operator sequences of the Tn10-encoded tetracycline resistance operon are determined in vitro by transcription studies of purified DNA restriction fragments, protection of guanosine from methylation by dimethylsulphate, and DNase I footprinting employing the purified TET repressor protein. In vitro transcription reveals three promoters with overlapping consensus sequences. Two of them, designated PR1 and PR2, are directed towards the tet repressor gene and the third, called PA, initiates transcription of the tet resistance gene. All three promoters are regulated simultaneously by the TET repressor protein, as demonstrated by in vitro transcription. Tetracycline functions as an inducer in these experiments. Two palindromic operator sequences in the tet operon control region, called O1 and O2, are occupied simultaneously by the TET repressor. Four guanosine residues in symmetric positions close to the centre of the palindromic operator sequences are protected from methylation in the repressor-operator complex. However, only one guanosine residue exhibits an enhanced reaction with dimethylsulphate under these conditions. Footprinting experiments reveal protection of phosphodiester bonds against DNase I slightly further than the palindromic sequence arrangement. Several phosphodiester bonds between the two operators are accessible for cleavage by DNase I in the repressor-operator complex. Two phosphodiester bonds within each operator sequence are cleaved by DNase I. This feature shows a clear assymmetry with the two inside cleavage positions of O1 and O2 being much less accessible for DNase I as compared to the two outside positions. A molecular mechanism of regulation of the Tn10-encoded tetracycline resistance operon is presented based on these and previous results.