James Tamara, Hsieh Meng-Lun, Knipling Leslie, Hinton Deborah
Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Building 8A, Room 2A13, 8 Center Drive, Bethesda, MD, 20892, USA.
Methods Mol Biol. 2015;1334:29-40. doi: 10.1007/978-1-4939-2877-4_3.
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.
确定蛋白质 - DNA 复合物的结构可能很困难，特别是当蛋白质与 DNA 的结合不紧密时，当没有可从中推断 DNA 结合情况的同源蛋白质时，和/或当只有部分蛋白质能够结晶时。如果蛋白质只是大型多亚基复合物的一部分，还可能出现其他复杂情况，例如复合物对于核磁共振研究来说太大，或者无法获得晶体学分析所需的蛋白质和核酸量。在这里，我们描述了一种用于绘制大型转录复合物中激活蛋白相对于 DNA 位置的技术。我们根据使用与化学切割试剂溴乙酰胺苄基 - 乙二胺四乙酸铁（FeBABE）在特定残基处偶联的激活蛋白生成的数据，确定了激活蛋白在 DNA 上的位置。这些分析与激活蛋白部分和 B 型 DNA 的可用结构的三维模型相结合，以获得蛋白质相对于 DNA 的三维图像。最后，使用 Molsoft 程序优化位置，揭示转录复合物中蛋白质 - DNA 的结构。
