Orientation and affinity of HIV-1 Tat fragments in Tat-TAR complex determined by fluorescence resonance energy transfer

Bioconjugate Chemistry
Hong CaoTariq M Rana

Abstract

The human immunodeficiency virus (HIV-1) encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis. Tat interacts with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Tat-derived peptides that contain the basic arginine-rich region of Tat are able to form in vitro complexes with TAR RNA, and these peptides provide a well-characterized system for understanding the mechanism of RNA-protein recognition. It is not known how RNA-binding Tat peptides are folded or docked in the Tat-TAR complex, and to what extent structural reorganization occurs upon TAR binding. To address these questions, we developed a fluorescence resonance energy transfer (FRET) system to analyze the interactions between TAR RNA and a Tat protein fragment (aa 38-72) uniquely labeled with donor and acceptor dye molecules, respectively. Using this FRET assay, we determined the binding affinity of Tat (47-58) and Tat (38-72) for TAR RNA under physiological conditions. We also delineated the distance between the N- and C-terminus of Tat (38-72) and the distance between the two termin...Continue Reading

References

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Nov 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·M G CordingleyA J Schlabach
Mar 3, 1999·Journal of Biomolecular Structure & Dynamics·M J SeewaldH Sticht
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Citations

Dec 5, 2006·Biochemical and Biophysical Research Communications·Yuhui MaJingquan Zhao
Dec 9, 2014·RNA Biology·Martyna O UrbanekWlodzimierz J Krzyzosiak
Nov 1, 2012·Journal of the American Chemical Society·Jaeyoung PaiInjae Shin
Dec 26, 2008·Bioorganic & Medicinal Chemistry Letters·Giuseppe ManfroniArnaldo Fravolini
Apr 28, 2007·Chemical Biology & Drug Design·Ya-Lin ChiuTariq M Rana

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