Open Access Dissertation
Date of Award
Professor Andrew L. Feig
Professor David S. Rueda
The Dimerization Initiation Sequence (DIS) is a conserved hairpin-loop motif on the 5' UTR of the HIV-1 genome. It plays an important role in genome dimerization through formation of a "kissing complex" intermediate between two homologous DIS sequences. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV. We wish to report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. Our work has employed single-molecule fluorescence resonance energy transfer to monitor the dimerization of minimal HIV-1 RNA sequence containing DIS. Most significantly, we observed a previously uncharacterized folding intermediate that plays a critical role in the dimerization mechanism. Our data clearly show that dimerization involves three distinct steps in dynamic equilibrium and regulated by Mg2+ ions. Two of the steps correspond to previously proposed structures: the kissing complex and the extended duplex. Surprisingly, our data reveal a previously unobserved obligatory folding intermediate, consistent with a bent kissing complex conformation, similar to the TAR complex. Mutations of the highly conserved purines flanking the DIS loop destabilize this intermediate, indicating that these purines may play an important role in the HIV-1 RNA dimerization in vivo.
Mundigala, Hansini R., "Hiv-1 Rna Dimerization At Single Molecule Level" (2014). Wayne State University Dissertations. 907.