Tzfira Tzvi, Citovsky Vitaly
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA.
Methods Mol Biol. 2008;451:293-316. doi: 10.1007/978-1-59745-102-4_21.
Most plant viruses move between plant cells with the help of their movement proteins (MPs). MPs are multifunctional proteins, and one of their functions is almost invariably binding to nucleic acids. Presumably, the MP-nucleic acid interaction is directly involved in formation of nucleoprotein complexes that function as intermediates in the cell-to-cell transport of many plant viruses. Thus, when studying a viral MP, it is important to determine whether or not it binds nucleic acids, and to characterize the hallmark parameters of such binding, i.e., preference for single- or double-stranded nucleic acids and binding cooperativity and sequence specificity. Here, we present two major experimental approaches, native gel mobility shift assay and ultra violet (UV) light cross-linking, for detection and characterization of MP binding to DNA and RNA molecules. We also describe protocols for purification of recombinant viral MPs over-expressed in bacteria and production of different DNA and RNA probes for these binding assays.
大多数植物病毒借助其运动蛋白（MPs）在植物细胞间移动。运动蛋白是多功能蛋白，其功能之一几乎总是与核酸结合。据推测，运动蛋白与核酸的相互作用直接参与核蛋白复合体的形成，这些复合体在许多植物病毒的细胞间运输中起中间体的作用。因此，在研究病毒运动蛋白时，确定它是否与核酸结合，并表征这种结合的标志性参数，即对单链或双链核酸的偏好、结合协同性和序列特异性，是很重要的。在这里，我们介绍两种主要的实验方法，即天然凝胶迁移率变动分析和紫外线（UV）交联，用于检测和表征运动蛋白与DNA和RNA分子的结合。我们还描述了在细菌中过表达的重组病毒运动蛋白的纯化方案，以及用于这些结合分析的不同DNA和RNA探针的制备方法。
