Serpa Viviane, Vernal Javier, Lamattina Lorenzo, Grotewold Erich, Cassia Raul, Terenzi Hernán
Laboratório de Expressão Gênica, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil.
Biochem Biophys Res Commun. 2007 Oct 5;361(4):1048-53. doi: 10.1016/j.bbrc.2007.07.133. Epub 2007 Jul 31.
Nitric oxide (NO) can influence the transcriptional activity of a wide set of Arabidopsis genes. The aim of the present work was to investigate if NO modifies DNA-binding activity of AtMYB2 (a typical R2R3-MYB from Arabidopsis thaliana), by a posttranslational modification of its conserved Cys53 residue. We cloned a fully active minimal DNA-binding domain of AtMYB2 spanning residues 19-125, hereafter called M2D. In EMSA assays, M2D binds the core binding site 5'-[A]AACC[A]-3'. The NO donors SNP and GSNO inhibit M2D DNA-binding. As expected for a Cys S-nitrosylation, the NO-mediated inhibitory effect was reversed by DTT, and S-nitrosylation of Cys53 in M2D was detected by biotin switch assays. These results demonstrate that the DNA-binding of M2D is inhibited by S-nitrosylation of Cys53 as a consequence of NO action, thus establishing for the first time a relationship between the redox state and DNA-binding in a plant MYB transcription factor.
一氧化氮（NO）能够影响拟南芥中一系列基因的转录活性。本研究的目的是探究NO是否通过对拟南芥典型的R2R3-MYB转录因子AtMYB2保守的Cys53残基进行翻译后修饰，来改变其DNA结合活性。我们克隆了AtMYB2一个具有完全活性的最小DNA结合结构域，该结构域跨越第19至125位氨基酸残基，以下称为M2D。在电泳迁移率变动分析（EMSA）实验中，M2D能结合核心结合位点5'-[A]AACC[A]-3'。NO供体硝普钠（SNP）和亚硝基谷胱甘肽（GSNO）可抑制M2D与DNA的结合。正如半胱氨酸S-亚硝基化所预期的那样，二硫苏糖醇（DTT）可逆转NO介导的抑制作用，并且通过生物素转换法检测到M2D中Cys53的S-亚硝基化。这些结果表明，由于NO的作用，Cys53的S-亚硝基化抑制了M2D与DNA的结合，从而首次在植物MYB转录因子中建立了氧化还原状态与DNA结合之间的关系。
