TET repressortetoperator complex formation induces conformational changes in thetetoperator DNA

Abstract

The structural changes of the tet operator DNA upon binding of the TET repressor protein are examined by circular dichroism. For this purpose a 70 bp DNA fragment was prepared which contains both tet operators. About 67% o'f the base pairs of this DNA are involved in specific interaction with the TET repressor.A rather large change in the CD of the DNA is induced by binding of the TET repressor. The shape of the CD difference spectrum is similar to the respective difference found for the lac operator DNA upon complex formation with the lac repressor. However, the effect induced by the TET repressor on tet operator DNA seems to comprise both the specific and non-specific effect of the lac repressor on the structure of DNA [Culard, F. and Maurizot, J.C. (1981) Nucl. Acids Res. 9, 5157-5184]. Specificity of binding is confirmed by the lack of any effect of the TET repressor on the CD of a 95 bp lac operator containing DNA fragment, by the reduced mobility of TET repressor-tet operator complexes on polyacrylamid gels under CD conditiones, and by a titration experiment of tet operator DNA with TET repressor employing the CD change. The latter experiment reveals a stoichiometry of four TET repressors per tet operon control region.

DNA structural deformations in the interaction of the controller protein C.AhdI with its operator sequence

“…cro repressor (26), the CD signal decreases on forming the DNA–protein complex. The observed increase in Δε in the C.AhdI tetrameric complex (from 2.9 to 5.2 cm −1  · M −1 per nucleotide) is amongst the largest observed, comparable but even larger than the increase in CD when two TET repressor dimers bind to the tet operator sequence (from 2.9 to 4.8 cm −1  · M −1 per nucleotide (27)). It is interesting to note that the bend angle (48°) introduced into DNA when a TET repressor dimer binds to its operator sequence (28) is almost identical to the bend angle (47°) when a C.AhdI dimer binds DNA, correlating with a similar increase in CD signal at 276 nm.…”

A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter

“…Protein was step-eluted at 250 mM imidazole. The concentrations of TetR and MerR were determined spectrophotometrically using an extinction coeYcient of 30,600 cm ¡1 M ¡1 [10], and 11,600 cm ¡1 M ¡1 [11], respectively.…”

A differential scanning calorimetry study of tetracycline repressor

“…After the purification procedure, the protein was highly pure (> 97%) as judged by SDS/PAGE and Coomassie Brilliant Blue staining. The dimer repressor concentration was determined spectrophotometrically using an excitation coefficient ε 280nm = 30 × 10 3 m −1 ·cm −1 [15]. The activity of the proteins was checked using the Tc titration method.…”