Methyl groups of thymine bases are important for nucleic acid recognition by DtxR
Methyl groups of thymine bases are important for nucleic acid recognition by DtxR
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DOI：
10.1021/bi0009284
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发表时间：
2000-08-29
期刊：
影响因子：
2.9
通讯作者：
Ringe, D
中科院分区：
文献类型：
--
作者：
Chen, CSY;White, A;Ringe, D
白喉毒素的表达受白喉毒素阻遏物（DtxR）控制。在高铁浓度的条件下，DtxR结合tox操纵子以抑制转录。为了研究如何通过这种阻遏物实现DNA结合特异性，我们解决了镍（II）激活的DtxR（C102 D）突变体与含有DtxR共有结合序列的43聚体DNA双链体复合的晶体结构。该复合物的结构分析和与先前确定的DtxR（C102 D）-Ni（II）-tox操作员三元复合物的比较揭示了阻遏物螺旋-转角-螺旋（HTH）基序的Ser 37/Pro 39与共有结合序列中特定胸腺嘧啶碱基的甲基之间的不寻常的货车德瓦耳斯相互作用。利用脱氧尿苷修饰的双链DNA探针的凝胶迁移率变动分析证明了这些相互作用的重要性：显示与晶体结构中的Ser 37/Pro39相互作用的四个甲基基团贡献了总共3.4千卡/摩尔的结合能。因此，除了通过其Gln 43残基与DNA进行碱基特异性氢键相互作用之外，DtxR还识别具有其Ser 37和Pro39侧链的DNA序列中某些位置的甲基，以实现对其同源操纵基因序列的结合特异性。
The expression of diphtheria toxin is controlled by the diphtheria toxin repressor (DtxR). Under conditions of high iron concentration, DtxR binds the tox operator to inhibit transcription. To study how DNA binding specificity is achieved by this repressor, we solved the crystal structure of the nickel(II) activated DtxR(C102D) mutant complexed with a 43mer DNA duplex containing the DtxR consensus binding sequence. Structural analysis of this complex and comparison with a previously determined DtxR(C102D)-Ni(II)-tox operator ternary complex revealed unusual van der Waals interactions between Ser37/Pro39 of the repressor helix-turn-helix (HTH) motif and the methyl groups of specific thymine bases in the consensus binding sequence. Gel mobility shift assays utilizing deoxyuridine modified duplex DNA probes proved the importance of these interactions: the four methyl groups shown to interact with Ser37/Pro39 in the crystal structure contribute a total of 3.4 kcal/mol to binding energy. Thus, in addition to making base-specific hydrogen-bonding interactions to the DNA through its Gln43 residue, DtxR also recognizes methyl groups at certain positions in the DNA sequence with its Ser37 and Pro39 side chains, to achieve binding specificity toward its cognate operator sequences.