Isothermal Calorimetric Studies of Arginine and Divalent Ion Binding to the TAR RNA

Abstract

The transactivation-response region (TAR) of HIV-1 increases its rate of transcription by one-hundred fold upon binding to the Tat protein. The TAR RNA of HIV-1 contains a conserved three nucleotide bulge: U23, C23, and U25; the crystal structure shows three calcium ions bound to the bulge. The base-triple, U23·A27-U38, is important for arginine binding (R52 of Tat). Thermal denaturation experiments on TAR RNA show that binding of divalent metal ions increases its stability relative to 1 M K+ ions. Isothermal Titration Calorimetry was used to measure the binding constant and enthalpy of binding for arginine and divalent metal ions. Arginine binds at one site on the TAR RNA whereas calcium and magnesium bind at a minimum of two sites. Calcium and magnesiumions also have different binding affinities for the two sites.