Poster abstracts
Poster number 23 submitted by Jonathan Kitzrow
Probing 5′UTR Conformational Dynamics by FRET
Jonathan P Kitzrow (Chemistry and Biochemistry at The Ohio State University), Benjamin Brigham (Molecular Biology & Microbiology at Tufts University), James Munro (Molecular Biology & Microbiology at Tufts University), Karin Musier-Forsyth (Chemistry and Biochemistry at The Ohio State University)
Abstract:
The full-length HIV-1 transcript can act as either mRNA, encoding the Gag and Gag-Pol polyproteins, or as genomic RNA (gRNA) that is selected by Gag during virion assembly. The HIV-1 5′UTR, which is part of all full-length HIV-1 transcripts, is known to regulate many important processes during the viral lifecycle, including RNA splicing, translation, dimerization, gRNA packaging, tRNA primer annealing and initiation of reverse transcription. The HIV-1 5′UTR is believed to exist on a structural tipping point between two predominant conformations, one in which the dimer initiation sequence (DIS) is exposed and available to dimerize and a second conformation in which the DIS is sequestered via interactions with upstream sequences. We have designed a FRET-based assay to investigate the gRNA 5′UTR conformation and dynamics using ensemble and single-molecule approaches. Our studies support the conclusion that the 5′UTR predominantly adopts two stable conformational states. Individual FRET traces showed that 29% of the molecules transitioned between states during the ~150 sec observation window. Inter-molecular FRET observed between an immobilized donor-labeled 5′UTR construct and excess acceptor-labeled 5′UTR was consistent with formation of an extended dimer. Interestingly, heat-annealing tRNALys3 to the primer binding site of an immobilized 5′UTR both in the absence and presence of excess free 5′UTR promoted the DIS sequestered monomeric conformation. In contrast, the presence of nucleocapsid protein (NC) in the absence of tRNA stabilizes high-FRET dimeric states. Overall, these data are consistent with high intrinsic conformational dynamics in the 5′UTR that can be regulated by tRNA primer annealing and NC.
Keywords: HIV-1 5UTR, RNA, FRET