Structural analyses of transcriptional regulation in stress response

TFIIE大亚基中的锌指结构域在系统发育上是保守的，对转录至关重要。我们已经确定了它的核磁共振结构。它由一个α螺旋和六个β链组成，显示出与先前确定的锌结合结构不同的新特征。我们在这个区域创建了点突变体，并检测了它们与其他一般转录因子的结合能力以及它们的转录活性。观察到Zn^<2+>-配体突变体有趣的功能不对称性：n端两个突变体在超卷曲模板上保持约20%的活性。CD和NMR研究表明，这两个突变体主要以随机线圈结构平衡，尽管四个突变体都具有部分折叠的特征结构，配位为Zn^<2+>-。此外，高度保守的d164突变体（D 164A和D 164K）也增加了转录。此外，我们确定了hTRF2与端粒双链DNA结合的DNA结合域的溶液结构，其序列为GTTAGGGTTAGGG，并与hTRF1相应的DNA复合体结构进行了比较。端粒是真核生物线性染色体的末端，由重复的富g序列和端粒重复结合因子组成。在哺乳动物端粒中，TRF1和TRF2与双链端粒DNA结合。两者都含有一个中心trf同源结构域和一个c端DNA结合结构域。两种dna结合结构彼此非常接近，然而，观察到微小但显著的结构差异。基于目前hTRF2的结构，我们可以制造出几种与端粒DNA结合能力比野生型更强的hTRF2突变体。

The zinc finger domain in the large subunit of TFIIE is phylogenetically conserved and is essential for transcription. We have determined its NMR structure. It consists of one alpha helix and six beta strands showing novel features distinct from previously determined zinc-binding structures. We created point mutants in this domain and examined their binding abilities to other general transcription factors as well as their transcription activities. Interesting functional asymmetry of Zn^<2+>-ligand mutants was observed: the N-terminal two mutants remained approximately 20% activity on a supercoiled template. CD and NMR studies showed that those two equilibrate mainly with the random coil structure, although all four mutants possessed partially folded characteristic structures coordinating Zn^<2+>-. And also, highly conserved D 164 mutants (D 164A and D 164K) were found to increase transcription.In addition, we have determined the solution structure of the DNA binding domain of hTRF2 bound to a telomeric double-stranded DNA with the sequence of GTTAGGGTTAGGG and compared it with the corresponding DNA complex structure of hTRF1. Telomeres are the ends of eukaryotic linear chromosomes consisting of repetitive G-rich sequence and telomeric repeat binding factors. In mammalian telomeres, TRF1 and TRF2 bind to double-stranded telomeric DNA. Both contain a central TRF-homology (TRFH) domain and a C-terminal DNA binding domain. Both DNA-bound structures are very close to each other, however, small but significant structural differences are observed. Based on the present structure of hTRF2 we could make several mutants of hTRF2, which have a stronger binding ability to telomeric DNA rather than the wild type.