Dissecting structural transitions in the HIV-1 dimerization initiation site RNA using 2-aminopurine fluorescence.

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TitleDissecting structural transitions in the HIV-1 dimerization initiation site RNA using 2-aminopurine fluorescence.
Publication TypeJournal Article
Year of Publication2009
AuthorsLee, H-W, Briggs, KT, Marino, JP
JournalMethods
Volume49
Issue2
Pagination118-27
Date Published2009 Oct
ISSN1095-9130
Keywords2-Aminopurine, Base Sequence, Binding Sites, Biophysics, Dimerization, gag Gene Products, Human Immunodeficiency Virus, HIV-1, Kinetics, Models, Chemical, Molecular Sequence Data, Nucleic Acid Conformation, Nucleocapsid, RNA, Viral, Spectrometry, Fluorescence, Thermodynamics
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.

DOI10.1016/j.ymeth.2009.05.006
Alternate JournalMethods
PubMed ID19460437
PubMed Central IDPMC2772140
Grant ListGM59107 / GM / NIGMS NIH HHS / United States
R01 GM059107-09 / GM / NIGMS NIH HHS / United States