Orientation and affinity of HIV-1 Tat fragments in Tat-TAR complex determined by fluorescence resonance energy transfer.
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 termini and the 5' end of TAR when Tat (38-72) is bound to TAR. Our results suggest that the N- and C-termini of Tat (38-72) are close to each other when the peptide is folded and that the peptide does not go through a large structural change upon TAR binding.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Hong
H
Cao
Natarajan
N
Tamilarasu
Tariq M
TM
Rana
D000595:Amino Acid Sequence; D001483:Base Sequence; D031541:Fluorescence Resonance Energy Transfer; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010446:Peptide Fragments; D054322:tat Gene Products, Human Immunodeficiency Virus