Global analysis of non-specific protein-nucleic interactions by sedimentation equilibrium.
Protein-nucleic acid interactions govern a variety of processes, including replication, transcription, recombination and repair. These interactions take place in both sequence-specific and non-specific modes, and the latter occur in many biologically significant contexts. Analytical ultracentrifugation is a useful method for the detailed characterization of the stoichiometry and affinity of macromolecular interactions in free solution. There has been a resurgence of interest in the application of sedimentation equilibrium methods to protein-nucleic acid interactions. However, these studies have been generally focused on sequence-specific interactions. Here we describe an approach to analyze non-specific interactions using sedimentation equilibrium. We have adapted an existing model for non-specific interaction of proteins with finite, one-dimensional nucleic acid lattices for global fitting of multiwavelength sedimentation equilibrium data. The model is extended to accommodate protein binding to multiple faces of the nucleic acid, resulting in overlap of consecutive ligands along the sequence of the RNA or DNA. The approach is illustrated in a sedimentation equilibrium analysis of the interaction of the double-stranded RNA binding motif of protein kinase R with a 20-basepair RNA construct.
Ucci
Jason W
JW
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Cole
James L
JL
D020029:Base Pairing; D008024:Ligands; D046911:Macromolecular Substances; D009696:Nucleic Acids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D014461:Ultracentrifugation / Q000379:methods*
D008024:Ligands; D046911:Macromolecular Substances; D009696:Nucleic Acids; D011506:Proteins; D012330:RNA, Double-Stranded; D012996:Solutions; D011494:Protein Kinases