Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program
Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program
复制标题
DOI：
10.1007/978-1-4939-2877-4_3
复制
发表时间：
2015-01-01
期刊：
影响因子：
--
通讯作者：
Hinton, Deborah
中科院分区：
文献类型：
--
作者：
James, Tamara;Hsieh, Meng-Lun;Hinton, Deborah
确定蛋白质-DNA复合物的结构可能是困难的，特别是如果蛋白质不与DNA紧密结合，如果不存在可以推断DNA结合的同源蛋白质，和/或如果只有部分蛋白质可以结晶。如果蛋白质仅包含大的多亚基复合物的一部分，则可能出现其他并发症，例如复合物对于NMR研究来说太大，或者不可能获得晶体学分析所需的蛋白质和核酸的量。在这里，我们描述了一种技术，我们用来映射的位置激活蛋白相对于一个大的转录复合物内的DNA。我们确定了激活剂的DNA上的位置，从使用激活剂蛋白，已在特定的残基与化学裂解试剂，铁溴乙酰氨基苄基-EDTA（FeBABE）共轭产生的数据。这些分析与激活蛋白和B型DNA部分的可用结构的3-D模型相结合，以获得蛋白质相对于DNA的3-D图像。最后，Molsoft程序用于细化位置，揭示转录复合物内蛋白质-DNA的结构。
Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex.