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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.

Human immunodeficiency virus, type 1 (HIV-1), Tat activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex, P-TEFb. Binding of Tat to CycT1 induces cooperative binding of the P-TEFb complex onto nascent HIV-1 TAR RNA. Here the specific interaction between Tat protein, human cyclin T1, and HIV-1 TAR RNA was analyzed by fluorescence resonance energy transfer, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein synthesized through solid-phase chemistry. We find that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA and that TAR RNA further enhances the interaction between Tat and CycT1. We conclude that TAR RNA nucleates the formation of the Tat·P-TEFb complex through an induced fit mechanism.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′-end of all HIV mRNAs. A number of cyclic peptides are known to possess antibiotic activity and increased biological stability. Here we report the design, synthesis, and biological activity of a cyclic peptide (2Figure 1The structure of tripeptide (1) and cyclic peptide (2).), which inhibits transcriptional activation by Tat protein in human cells with an IC50of ≈40 nM. Cyclic peptides that can target specific RNA structures provide a new class of small molecules that can be used to control cellular processes involving RNA–protein interactions in vivo.

The chemoselective transformation of Trp 9and Trp 11, located at the hydrophobic core of the antiparallel, double helical pore of O-acetyl gramicidin A(HCO-ValGlyAlaLeu oAlaVal oValVal oTrpLeu oTrpLeu oTrpLeu oTrpNHCH 2CH 2OAc; o = D isomer) to Asp 9gramicidin and Asp 9Asp 11gramicidin has been demonstrated by oxidation with 0.25 eq. of in situgenerated Ru(VIII), HPLC isolation, amino acid analysis and peptide sequencing.

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