Poster abstracts

Poster number 49 submitted by Erik Olson

Sample Preparation and SAXS analyses of RNAs derived from the 5´UTR of the HIV-1 genome

Erik David Olson (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Chris P. Jones (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210)

Abstract:
The 5´ untranslated region (5´UTR) of the human immunodeficiency virus type 1 (HIV-1) RNA genome (vRNA) controls virus replication via its rich secondary structure. For example, the dimer initiation sequence (DIS) and Psi hairpins control the coordinated process of vRNA dimerization and packaging. While the secondary structure of the 5´UTR is well annotated, a model for the tertiary structure of the entire 5´UTR remains elusive. The overall goal of this project is to elucidate the global fold of various RNA constructs derived from the 5´UTR of the vRNA using small angle X-ray scattering (SAXS). The 5´UTR is hypothesized to be a riboswitch that exists in a monomeric state in its role in viral protein translation and in a dimeric state that promotes packaging and virus assembly. Initial structural studies are being performed on monomeric 5´UTR constructs in which the DIS has been mutated to prevent dimerization. SAXS requires homogeneous sample preparation, which we accomplished through optimization of construct design and RNA refolding, and purification via size-exclusion chromatography (SEC). We show that by replacing divalent Mg²⁺ cations with monovalent K⁺ cations during folding of the 5´UTR constructs, sample homogeneity was increased. Additionally, by removing Mg²⁺ from the SEC/storage buffer, the 5´ UTR constructs remain stably monomeric at 4˚C after SEC purification. These optimization steps have yielded 5´UTR RNA constructs that are amenable to SAXS analysis and the results of initial SAXS studies will be presented. This work represents an important step towards building a low-resolution model of the entire 5´UTR in the absence and presence of interacting partners.

Keywords: HIV-1, SAXS, 5UTR