title: Studying the affinity, kinetic stability, and specificity of RNA/protein interactions: SINE ncRNA/Pol II complexes as a model system.
abstract: The number of documented interactions between proteins and noncoding RNAs (ncRNA) of all types has grown rapidly in the past several years. A current challenge is to experimentally characterize these interactions to ultimately understand their biological roles at a mechanistic level, which will require a combination of multiple experimental techniques. One such category of techniques is biochemical assays that determine the affinity, kinetic stability, and specificity of ncRNA/protein complexes. Here we describe how to experimentally determine these important parameters using electrophoretic mobility shift assays (EMSAs). The interaction between mammalian SINE-encoded ncRNAs and human RNA polymerase II is presented as a model system; however, the experiments could be readily adapted to other ncRNA/protein complexes.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, 596 UCB, Boulder, CO, 80309-0215, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Goodrich
authors.forename: Jennifer F
authors.initials: JF
authors.lastname: Kugel
mesh_terms: D001667:Binding, Competitive; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006801:Humans; D007700:Kinetics; D012319:RNA Polymerase II / Q000378:metabolism*; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*
chemical_list: D022661:RNA, Untranslated; D012319:RNA Polymerase II