Expression, purification and operator binding of the transposon Tn1721-encoded Tet repressor

The regulation of expression of the Tn1721-encoded tetracycline-resistance determinant is described at the molecular level. The transcriptional control element consists of overlapping divergent promoters, which are negatively regulated by two operators with nearly identical sequence. The mRNA for the regulatory gene tetR is translated without a ribosome-binding site. This result is confirmed by S1 nuclease mapping and RNA sequencing of the tetR mRNA. The start nucleotide for transcription of this mRNA is the adenosine residue of the sequence 5'-AUG. Determination of the N-terminal amino acid sequence of the purified Tet repressor proves that this AUG is the initiation codon for translation. The Tet repressor protein is further used to map the two tet operators by DNase I footprinting. Tight contacts of the protein to the N-7 positions of two guanosine residues in each operator are determined from methylation protection experiments with dimethylsulfate. The differential regulation and positive control of transcription of the tetR gene that is possible with this arrangement of promoters and operators is discussed.