DISSECTION OF THE DNA-BINDING DOMAIN OF XENOPUS-LAEVIS TFIIIA - QUANTITATIVE DNASE-I FOOTPRINTING ANALYSIS OF SPECIFIC COMPLEXES BETWEEN A 5-S RNA GENE FRAGMENT AND N-TERMINAL FRAGMENTS OF TFIIIA CONTAINING 3-ZINC-FINGER, 4-ZINC-FINGER OR 5-ZINC-FINGER DOMAINS
DISSECTION OF THE DNA-BINDING DOMAIN OF XENOPUS-LAEVIS TFIIIA - QUANTITATIVE DNASE-I FOOTPRINTING ANALYSIS OF SPECIFIC COMPLEXES BETWEEN A 5-S RNA GENE FRAGMENT AND N-TERMINAL FRAGMENTS OF TFIIIA CONTAINING 3-ZINC-FINGER, 4-ZINC-FINGER OR 5-ZINC-FINGER DOMAINS
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DOI：
10.1006/jmbi.1993.1499
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发表时间：
1993-09-20
影响因子：
5.6
通讯作者：
THOGERSEN, HC
中科院分区：
文献类型：
--
作者：
HANSEN, PK;CHRISTENSEN, JH;THOGERSEN, HC
由大肠杆菌可清除的杂合蛋白产生的非洲爪蟾转录因子Ⅲ A（TFIIIA）的N端片段所对应的重组锌指蛋白与含有5S RNA基因内部控制区（ICR）的DNA片段形成特异性的化学计量复合物。3种蛋白质在ICR上的DNA酶I足迹的有序集合包括从其3′端（相对于成熟转录物起始的+96位置）分别向上游延伸20 bp、20 bp或34 bp进入ICR的嵌套足迹集合。足迹数据的定量分析提供了有力的证据，证明DNA酶I足迹，因此结构，真正的TFIIIA：该区域中的ICR复合物完全和精确地由N-末端三指结合在+77至+96区域内加上一对指4和5来解释，都需要将足迹从+77向上延伸到+63位置。根据先前的结果和数据，提出并讨论了这组新足迹数据的结构解释，其中蛋白质-ICR和三个N-末端指之间的DNA相互作用与在同源三指zif 268：DNA复合物中观察到的相互作用密切对应，而蛋白质-DNA相互作用的基本模式（其中一对指4和5参与形成TFIIIA：ICR复合物）具有完全不同的性质，尽管尚未理解。为了评估我们的模型在潜在的特异性决定H-键合模式，三指TFIIIA片段和ICR之间的复合物的分子模型构建，使用zif 268：DNA坐标。根据我们的模型，九个氨基酸残基中有八个适合与碱基形成氢键，是潜在的氢键受体或供体。
Recombinant zinc finger proteins corresponding to N-terminal fragments ofXenopus laevistranscription factor IIIA (TFIIIA) comprising three, four and five fingers produced inEscherichia colias clearable hybrid proteins were shown to form specific stoichiometric complexes with DNA fragments containing the internal control region (ICR) of a 5 S RNA gene. The ordered set of DNase I footprints of each of the three proteins on the ICR comprise a nested set of footprints extending upstream from its 3′ end (position +96 relative to start of the mature transcript) 20 bp, 20 bp or 34 bp into the ICR, respectively. Quantitative analysis of the footprinting data provided firm evidence that the DNase I footprint, and hence the structure, of the authentic TFIIIA : ICR complex in this region is fully and precisely accounted for by the N-terminal three fingers binding within the +77 to +96 region plus the pair of fingers 4 and 5, both required to extend the footprint upwards from the +77 to the +63 position.A structural interpretation of this set of new footprinting data in view of previous results and data is presented and discussed in terms of a refined model in which the protein-DNA interaction between the ICR and the three N-terminal fingers corresponds closely to that observed in the homologous three finger zif268 : DNA complex, whereas the basic mode of protein-DNA interaction, in which the pair of fingers 4 and 5 is engaged in forming the TFIIIA : ICR complex is of an entirely different, albeit not yet understood nature. To allow assessment of our model in terms of potential specificity-determining H-bonding patterns, a molecular model of the complex between the three finger TFIIIA fragment and the ICR was constructed, using the zif268 : DNA co-ordinates. Eight out of the nine amino acid residues, which according to our model are suitably located for forming hydrogen bonds with the bases, are potential H-bond acceptors or donors.