Thermodynamic Analysis of Tetracycline-Mediated Induction of Tet Repressor by a Quantitative Methylation Protection Assay
✍ Scribed by Thomas Lederer; Masayuki Takahashi; Wolfgang Hillen
- Publisher
- Elsevier Science
- Year
- 1995
- Tongue
- English
- Weight
- 160 KB
- Volume
- 232
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
been extensively studied in recent years, thermody-We describe a method for quantitative detection and namic description of the induction process involves a thermodynamic interpretation of tetracycline (tc)-medicomplicated scheme of coupled reactions which is not ated induction of the Tn10 encoded Tet repressor (TetR). yet understood.
Binding of dimeric TetR to the tet operator (tetO) was
In the past, footprinting techniques like DNase I footquantitated by protection of DNA from methylation as printing (13) and modification interference or proteca function of tc concentration. A thermodynamic scheme tion studies ( 14) have been widely used to characterize covering all single reactions relevant for TetR induction protein-DNA interactions (15,16). Quantitative footwas used to interpret the data. The equilibrium associaprinting methods have been established for thermodytion constants of the TetR-[Mg-tc] / and TetR-[Mg-tc] / 2 namic analysis (17). We describe here a quantitative complexes to tetO were determined at different NaCl methylation protection assay to analyze the thermodyand TetR concentrations. Variation of total TetR concennamic parameters of induction of Tn10-encoded TetR tration from 0.2 to 1.1 1 10 07 M yielded identical results. by tetracycline (tc). The dimeric TetR binds the tet oper-A strong salt dependency of TetR-tetO binding was veriator (tetO), resulting in protection of two G residues fied between 2.5 and 100 mM NaCl, whereas [Mg-tc] / from methylation (18). The inducer tc forms a [Mgbinding to TetR is independent of the ionic strength. The tc] / complex which binds with high affinity (K A É 10 9 TetR-tetO binding constant drops 10 2 -to 10 3 -fold upon M 01 ) (6) to the repressor-operator complex, leading to binding of the first and further 10 4 -to 10 7 -fold by binding reduced operator affinity and, hence, deprotection of of the second [Mg-tc] / . This apparent cooperativity of the G residues (18). We devised a scheme of coupled tc-mediated induction indicates that each [Mg-tc] / inequilibria to thermodynamically interpret the inducteracts with both TetR monomers.