Identification of a consensus DNA-binding site for the Arabidopsis thaliana SBP domain transcription factor, AtSPL14, and binding kinetics by surface plasmon resonance
Identification of a consensus DNA-binding site for the Arabidopsis thaliana SBP domain transcription factor, AtSPL14, and binding kinetics by surface plasmon resonance
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DOI：
10.1021/bi701431y
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发表时间：
2008-03-25
期刊：
影响因子：
2.9
通讯作者：
Stone, Julie M.
中科院分区：
文献类型：
--
作者：
Liang, Xinwen;Nazarenus, Tara J.;Stone, Julie M.
具有保守的富含Cys和his的SQUAMOSA启动子结合蛋白（SBP）结构域的蛋白质是具有新型双锌指结构域的光合生物的转录因子。尽管一些SBP编码基因的表达改变对生物体的生长发育有深远的影响，但对SBP结构域蛋白靶基因的了解甚少。拟南芥AtSPL14 SBP结构域基因的错误表达可以抵抗细胞程序性死亡并改变植物结构。通过配体的系统进化，通过指数富集（SELEX）或随机结合位点选择（RBSS）确定了AtSPL14的一致dna结合基序。AtSPL14识别的DNA包含其他SBP结构域蛋白的核心结合基序（GTAC），但突变分析表明，至少一个额外的侧翼核苷酸是有效的AtSPL14-DNA相互作用所必需的。几种SBP结构域氨基酸序列的比较使我们能够假设哪些特定的氨基酸可能参与这种序列特异性的DNA识别。突变体AtSPL14 DNA结合结构域蛋白的电泳迁移转移试验（EMSA）表明，并非所有的第二Zn结构中的Zn2+离子配位体都是DNA结合所严格要求的。采用表面等离子体共振（SPR）评价AtSPL14体外结合动力学，比较其与其他SBP结构域蛋白的平衡结合常数。这些数据为进一步的实验提供了强有力的基础，旨在定义和区分密切相关的SBP结构域家族成员所调节的基因集。
Proteins with a conserved Cys- and His-rich SQUAMOSA promoter binding protein (SBP) domain are transcription factors restricted to photosynthetic organisms that possess a novel two Zn-finger structure DNA-binding domain. Despite the fact that altered expression of some SBP-encoding genes has profound effects on organism growth and development, little is known about SBP domain protein target genes. Misexpression of the Arabidopsis thaliana AtSPL14 SBP domain gene confers resistance to programmed cell death and modifies plant architecture. A consensus DNA-binding motif for AtSPL14 was identified by systematic evolution of ligands by exponential enrichment (SELEX) or random binding site selection (RBSS). DNA recognized by AtSPL14 contained the core binding motif (GTAC) found for other SBP domain proteins, but mutational analyses indicated that at least one additional flanking nucleotide is necessary for effective AtSPL14-DNA interaction. Comparison of several SBP domain amino acid sequences allows us to hypothesize which specific amino acids might participate in this sequence-specific DNA recognition. Electrophoretic mobility shift assays (EMSA) with mutant AtSPL14 DNA-binding domain proteins indicated that not all of the Zn2+ ion coordinating ligands in the second Zn structure are strictly required for DNA binding. Surface plasmon resonance (SPR) was used to evaluate AtSPL14 in vitro binding kinetics for comparison of equilibrium binding constants with other SBP domain proteins. These data provide a strong basis for further experiments aimed at defining and distinguishing the sets of genes regulated by the closely related SBP domain family members.