Muhl Daniela, Jessberger Nadja, Hasselt Kristin, Jardin Christophe, Sticht Heinrich, Burkovski Andreas
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
BMC Mol Biol. 2009 Jul 23;10:73. doi: 10.1186/1471-2199-10-73.
The TetR family member AmtR is the central regulator of nitrogen starvation response in Corynebacterium glutamicum. While the AmtR regulon was physiologically characterized in great detail up to now, mechanistic questions of AmtR binding were not addressed. This study presents a characterization of functionally important amino acids in the DNA binding domain of AmtR and of crucial nucleotides in the AmtR recognition motif.
Site-directed mutagenesis, the characterization of corresponding mutant proteins by gel retardation assays and surface plasmon resonance and molecular modelling revealed several amino acids, which are directly involved in DNA binding, while others have more structural function. Furthermore, we could show that the spacing of the binding motif half sites is crucial for repression of transcription by AmtR.
Although the DNA binding domain of TetR-type repressors is highly conserved and a core binding motif was identified for AmtR and TetR(D), the AmtR binding domain shows individual properties compared to other TetR proteins. Besides by distinct amino acids of AmtR, DNA binding is influenced by nucleotides not only of the conserved binding motif but also by spacing nucleotides in C. glutamicum.
TetR家族成员AmtR是谷氨酸棒杆菌氮饥饿反应的核心调节因子。虽然到目前为止，AmtR调控子已在生理学上得到了详细表征，但尚未解决AmtR结合的机制问题。本研究对AmtR DNA结合结构域中功能重要的氨基酸以及AmtR识别基序中的关键核苷酸进行了表征。
定点诱变、通过凝胶阻滞分析、表面等离子体共振和分子建模对相应突变蛋白进行表征，揭示了几个直接参与DNA结合的氨基酸，而其他氨基酸具有更多的结构功能。此外，我们可以证明结合基序半位点的间距对于AmtR抑制转录至关重要。
虽然TetR型阻遏物的DNA结合结构域高度保守，并且已鉴定出AmtR和TetR(D)的核心结合基序，但与其他TetR蛋白相比，AmtR结合结构域具有个体特性。除了AmtR的不同氨基酸外，DNA结合不仅受保守结合基序核苷酸的影响，还受谷氨酸棒杆菌中间距核苷酸的影响。
