Schneider T L, Schepartz A
Department of Chemistry, Yale University, New Haven, Connecticut 06511, USA.
Biochemistry. 2001 Mar 6;40(9):2835-43. doi: 10.1021/bi002450+.
The rapid and correct assembly of dimeric transcription factors on target DNA is essential for accurate transcriptional regulation. Here we ask how a viral accessory factor, hepatitis B virus X protein (pX), influences the rate and identity of the assembly pathway followed by members of the basic region leucine zipper (bZIP) transcription factor family. A combination of fluorescence polarization and fluorescence resonance energy transfer (FRET) experiments demonstrates unequivocally that pX does not increase the concentration of properly folded bZIP dimers in solution. Rather, fluorescence polarization and gel mobility shift experiments reveal that pX interacts directly with the basic-spacer segment of the bZIP peptide and stabilizes the complex composed of this monomer and target DNA. By stabilizing the intermediate formed along the monomer assembly pathway but not the one formed along the dimer pathway, pX enhances the equilibrium stability of a bZIP.DNA complex without changing the molecular mechanism used for complexation. Additional experiments reveal that pX decreases the kinetic specificity of certain bZIP proteins. To the extent that it is reflected at the transcriptional level, this loss in specificity could have far-reaching consequences for the host cell.
二聚体转录因子在靶DNA上的快速正确组装对于精确的转录调控至关重要。在此，我们探讨病毒辅助因子乙型肝炎病毒X蛋白（pX）如何影响碱性区域亮氨酸拉链（bZIP）转录因子家族成员所遵循的组装途径的速率和特性。荧光偏振和荧光共振能量转移（FRET）实验相结合，明确证明pX不会增加溶液中正确折叠的bZIP二聚体的浓度。相反，荧光偏振和凝胶迁移率变动实验表明，pX直接与bZIP肽的碱性间隔区相互作用，并稳定由该单体和靶DNA组成的复合物。通过稳定沿单体组装途径形成的中间体而非沿二聚体途径形成的中间体，pX增强了bZIP-DNA复合物的平衡稳定性，而不改变用于形成复合物的分子机制。额外的实验表明，pX降低了某些bZIP蛋白的动力学特异性。就其在转录水平上的反映而言，这种特异性的丧失可能对宿主细胞产生深远影响。
