Probing interactions between plant virus movement proteins and nucleic acids.
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.
Tzfira
Tzvi
T
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA.
Citovsky
Vitaly
V
D002021:Buffers; D002498:Centrifugation / Q000379:methods; D004279:DNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D007202:Indicators and Reagents; D007700:Kinetics; D053830:Plant Viral Movement Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010942:Plant Viruses / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D002021:Buffers; D004279:DNA, Viral; D007202:Indicators and Reagents; D053830:Plant Viral Movement Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins