Poster abstracts

Poster number 55 submitted by Charles Medert

Characterization of hairpin elements in the HIV splice site A7 through isotopic segmental labeling

Charles M. Medert (Department of Chemistry and Biochemistry, Miami University), Jeffrey D. Levengood (Department of Chemistry and Biochemistry, Miami University)

Abstract:
The HIV splice site A7 (ssA7) dynamically associates with the competing host proteins heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and alternative splicing factor (ASF) to regulate splicing activity in a down-regulatory and up-regulatory fashion, respectively. Hitherto, these associations have been described according to ssA7 secondary structure and specific protein binding sites within the construct. The mechanism by which ssA7 recruits these host proteins, however, is not well understood and may be a consequence of synergistic associations between hairpin elements within ssA7 that prompt a change in protein affinity. Thus elucidating three-dimensional structural characteristics using high-resolution NMR spectroscopy may enhance our appreciation for how HIV splicing mechanics operate. However, due to the inherent limitations of NMR analysis for large nucleic acid constructs, a segmental isotopic labeling approach was taken for studying the first stem loop (SL1) of ssA7. Herein, we engineered a restriction site within ssA7 for the endonuclease MSL1 that enabled linearization of the plasmid and subsequent translation of homogenous SL1 for later use in multi-dimensional NMR studies. Concurrently, a trans-acting hammerhead ribozyme was made to induce site-specific phosphodiester cleavage yielding an unlabeled SL2,SL3 construct. Three dimensional information for SL1 in context of its tertiary interactions with SL2,SL3 could then be collected after ligation of the two constructs. Hence, understanding the difference between free SL1 structure and SL1 in the context of ssA7 may provide insight into the likely tertiary interactions.

Keywords: HIV, Splicing, NMR