title: Binding by TRBP-dsRBD2 Does Not Induce Bending of Double-Stranded RNA.
abstract: Protein-nucleic acid interactions are central to a variety of biological processes, many of which involve large-scale conformational changes that lead to bending of the nucleic acid helix. Here, we focus on the nonsequence-specific protein TRBP, whose double-stranded RNA-binding domains (dsRBDs) interact with the A-form geometry of double-stranded RNA (dsRNA). Crystal structures of dsRBD-dsRNA interactions suggest that the dsRNA helix must bend in such a way that its major groove expands to conform to the dsRBD's binding surface. We show through isothermal titration calorimetry experiments that dsRBD2 of TRBP binds dsRNA with a temperature-independent observed binding affinity (KD ∼500 nM). Furthermore, a near-zero observed heat capacity change (ΔCp = 70 ± 40 cal·mol(-1)·K(-1)) suggests that large-scale conformational changes do not occur upon binding. This result is bolstered by molecular-dynamics simulations in which dsRBD-dsRNA interactions generate only modest bending of the RNA along its helical axis. Overall, these results suggest that this particular dsRBD-dsRNA interaction produces little to no change in the A-form geometry of dsRNA in solution. These results further support our previous hypothesis, based on extensive gel-shift assays, that TRBP preferentially binds to sites of nearly ideal A-form structure while being excluded from sites of local deformation in the RNA helical structure. The implications of this mechanism for efficient micro-RNA processing will be discussed.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania.
authors.forename: Roderico
authors.initials: R
authors.lastname: Acevedo
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania.
authors.forename: Declan
authors.initials: D
authors.lastname: Evans
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania.
authors.forename: Katheryn A
authors.initials: KA
authors.lastname: Penrod
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania. Electronic address: sas76@psu.edu.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Showalter
mesh_terms: D002151:Calorimetry; D006801:Humans; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D000072417:Protein Domains; D012330:RNA, Double-Stranded / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012996:Solutions; D013696:Temperature; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D012996:Solutions; D000099253:Trans-Activation Responsive RNA-Binding Protein