Poster abstracts
Poster number 29 submitted by Jonathan P. Kitzrow
Impact of transcription start site heterogeneity on HIV-1 5′UTR structure and conformational dynamics
Jonathan P. Kitzrow (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Shuohui Liu (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Kevin Jamison (Department of Physics, The Ohio State University, Columbus, OH 43210), Dennis Bong (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Michael Poirier (Department of Chemistry and Biochemistry, Center for RNA Biology, Department of Physics, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)
Abstract:
The highly conserved ~350-nt 5′UTR of the HIV-1 RNA genome (gRNA) is central to the regulation of virus replication. Interactions between the 5′UTR and viral and host factors are critical to the production of infectious virions. Previous biochemical and NMR experiments support a model in which the 5′UTR can adopt at least two mutually exclusive conformational states. In one state, the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed and the genome is competent for dimerization and packaging into assembling virions. We have previously characterized the intrinsic conformational dynamics of a 238-nt 5′UTR lacking the 5′ TAR/PolyA hairpin domains using single-molecule Förster resonance energy transfer (FRET). We showed that viral and host factor binding modulates the RNA conformation and dynamics. Recently, heterogenous transcription start site selection has been implicated in the localization of HIV-1 gRNA; transcripts with three 5′ guanosines (3G) are abundant in the cytoplasm while 1G transcripts are packaged into virions. Here, we investigate the structure and conformational dynamics of the full 1G and 3G 5′UTRs using a new bifacial peptide nucleic acid-labeling strategy to position internal fluorophores. In-gel selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) and FRET support the conclusion that 1G and 3G 5′UTRs adopt different conformations. Single-molecule FRET studies to probe the conformational dynamics of 1G/3G RNAs are underway. This work shows how a 2-nt sequence difference within a 9.4 kb HIV-1 gRNA can shift the global 5′UTR conformation, and supports the development of RNA-binding therapeutics that can shift the equilibrium to a non-functional state.
Keywords: HIV-1, RNA Structure, FRET