Phillips Nelson B, Jancso-Radek Agnes, Ittah Varda, Singh Rupinder, Chan Ging, Haas Elisha, Weiss Michael A
Department of Biochemistry, Case School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935, USA.
J Mol Biol. 2006 Apr 21;358(1):172-92. doi: 10.1016/j.jmb.2006.01.060. Epub 2006 Feb 6.
Human testis-determining factor SRY contains a high-mobility-group (HMG) box, an alpha-helical DNA-binding domain that binds within an expanded minor groove to induce DNA bending. This motif is flanked on the C-terminal end by a basic tail, which functions both as a nuclear localization signal and accessory DNA-binding element. Whereas the HMG box is broadly conserved among otherwise unrelated transcription factors, tails differ in sequence and mode of DNA binding. Contrasting examples are provided by SRY and lymphoid enhancer factor 1 (LEF-1): whereas the SRY tail remains in the minor groove distal to the HMG box, the LEF-1 tail binds back across the center of the bent DNA site. The LEF-1 tail relieves electrostatic repulsion that would otherwise be incurred within the compressed major groove to enable sharp DNA bending with high affinity. Here, we demonstrate that the analogous SRY tail functions as a "kinetic clamp" to regulate the lifetime of the bent DNA complex. As in LEF-1, partial truncation of the distal SRY tail reduces specific DNA affinity and DNA bending, but these perturbations are modest: binding is reduced by only 1.8-fold, and bending by only 7-10 degrees . "Tailed" and truncated SRY complexes exhibit similar structures (as probed by NMR) and distributions of long-range conformational substates (as probed by time-resolved fluorescence resonance energy transfer). Surprisingly, however, the SRY tail retards dissociation of the protein-DNA complex by 20-fold. The marked and compensating changes in rates of association and dissociation observed on tail truncation, disproportionate to perturbations in affinity or structure, suggest that this accessory element functions as a kinetic clamp to regulate the lifetime of the SRY-DNA complex. We speculate that the kinetic stability of a bent DNA complex is critical to the assembly and maintenance of a sex-specific transcriptional pre-initiation complex.
人类睾丸决定因子SRY包含一个高迁移率族（HMG）盒，这是一个α螺旋DNA结合结构域，它在扩展的小沟内结合以诱导DNA弯曲。该基序在C末端侧翼有一个碱性尾巴，其功能既是核定位信号又是辅助DNA结合元件。虽然HMG盒在其他不相关的转录因子中广泛保守，但尾巴在DNA结合序列和模式上有所不同。SRY和淋巴样增强子因子1（LEF-1）提供了对比实例：SRY尾巴保留在HMG盒远端的小沟中，而LEF-1尾巴则反向结合在弯曲DNA位点的中心。LEF-1尾巴减轻了在压缩的大沟内否则会产生的静电排斥，从而能够以高亲和力实现尖锐的DNA弯曲。在这里，我们证明类似的SRY尾巴作为“动力学钳”来调节弯曲DNA复合物的寿命。与LEF-1一样，远端SRY尾巴的部分截短会降低特定DNA亲和力和DNA弯曲，但这些扰动较小：结合仅降低1.8倍，弯曲仅降低7 - 10度。“有尾巴的”和截短的SRY复合物表现出相似的结构（通过核磁共振探测）和长程构象亚状态分布（通过时间分辨荧光共振能量转移探测）。然而，令人惊讶的是，SRY尾巴使蛋白质 - DNA复合物的解离延迟了20倍。在尾巴截短后观察到的结合和解离速率的显著且补偿性变化，与亲和力或结构的扰动不成比例，表明这个辅助元件作为动力学钳来调节SRY - DNA复合物的寿命。我们推测弯曲DNA复合物的动力学稳定性对于性别特异性转录预起始复合物的组装和维持至关重要。
