Abstract
A highly conserved 35 nucleotide RNA stem-loop, the dimerization initiation site (DIS), in the 5′ untranslated region (UTR) of the human immunodeficiency virus type I (HIV-1) genome has been identified as the sequence primarily responsible for initiation of viral genome dimerization. The DIS initiates viral genome dimerization through a loop-loop ‘kissing’ interaction and is converted from an intermediate ‘kissing’ to a more thermodynamically stable extended duplex dimer in a conformational rearrangement that is chaperoned by the HIV-1 nucleocapsid protein (NCp7). Here we describe fluorescence methods designed to probe local RNA dynamics and structural transitions associated with the DIS dimer formation and its NCp7 chaperoned structural conversion. These methods take advantage of the exquisite sensitivity of the quantum yield of the fluorescent nucleotide base analog, 2-aminopurine (2-AP), to its immediate structural and dynamic environment. The 2-AP fluorescence methods described allow a detailed kinetic and thermodynamic examination of this type of RNA-RNA interaction, as well as an analysis of the molecular mechanism of NCp7 chaperone activity.
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