Burgos Zepeda Monica Y, Alessandri Kevin, Murat Dorothée, El Amri Chahrazade, Dassa Elie
Unité des Membranes Bactériennes-CNRS URA2172, Département de Microbiologie, Institut Pasteur, 75724 Paris Cedex 15, France.
Biochim Biophys Acta. 2010 Apr;1804(4):755-61. doi: 10.1016/j.bbapap.2009.11.017. Epub 2009 Nov 27.
The Uup protein belongs to a subfamily of soluble ATP-binding cassette (ABC) ATPases that have been implicated in several processes different from transmembrane transport of molecules, such as transposon precise excision. We have demonstrated previously that Escherichia coli Uup is able to bind DNA. DNA binding capacity is lowered in a truncated Uup protein lacking its C-terminal domain (CTD), suggesting a contribution of CTD to DNA binding. In the present study, we characterize the role of CTD in the function of Uup, on its overall stability and in DNA binding. To this end, we expressed and purified isolated CTD and we investigated the structural and functional role of this domain. The results underline that CTD is essential for the function of Uup, is stable and able to fold up autonomously. We compared the DNA binding activities of three versions of the protein (Uup, UupDeltaCTD and CTD) by an electrophoretic mobility shift assay. CTD is able to bind DNA although less efficiently than intact Uup and UupDeltaCTD. These observations suggest that CTD is an essential domain that contributes directly to the DNA binding ability of Uup.
Uup蛋白属于可溶性ATP结合盒（ABC）ATP酶的一个亚家族，这些ATP酶参与了一些与分子跨膜运输不同的过程，如转座子精确切除。我们之前已经证明大肠杆菌Uup能够结合DNA。在缺乏其C末端结构域（CTD）的截短Uup蛋白中，DNA结合能力降低，这表明CTD对DNA结合有贡献。在本研究中，我们表征了CTD在Uup功能、其整体稳定性以及DNA结合中的作用。为此，我们表达并纯化了分离的CTD，并研究了该结构域的结构和功能作用。结果强调CTD对Uup的功能至关重要，它是稳定的且能够自主折叠。我们通过电泳迁移率变动分析比较了该蛋白的三个版本（Uup、UupDeltaCTD和CTD）的DNA结合活性。CTD能够结合DNA，尽管效率低于完整的Uup和UupDeltaCTD。这些观察结果表明CTD是一个直接有助于Uup DNA结合能力的必需结构域。
