P rotein-nucleic acid interactions are involved in many cellular functions, including transcription, RNA splicing, and translation. Readily accessible synthetic molecules that can bind with high affinity to specific sequences of single-or double-stranded nucleic acids have the potential to interfere with these interactions in a controllable way, making them attractive tools for molecular biology and medicine. Successful approaches used thus far include duplex-forming (antisense) (1) and triplexforming (antigene) oligonucleotides (2-4), peptide nucleic acids (5), and pyrrole-imidazole polyamide oligomers (6, 7). Each class of compounds uses a readout system based on simple rules for recognizing the primary or secondary structure of a linear nucleic acid sequence. Another approach uses carbohydratebased ligands, calicheamicin oligosaccharides, which interfere with the sequence-specific binding of transcription factors to DNA and inhibit transcription in vivo (8, 9). Although antisense oligonucleotides and peptide nucleic acids use the familiar Watson-Crick base-pairing rules, two others, the triplex-forming oligonucleotides and the pyrrole-imidazole polyamides, take advantage of straightforward rules to read the major and minor grooves, respectively, of the double helix itself.

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 Tat⅐P-TEFb complex through an induced fit mechanism. The human immunodeficiency virus (HIV-1)1 encodes a transcriptional activator protein, Tat, that increases the processivity of RNA polymerase II (for reviews see Refs. 1-3). Tat activates transcription through binding to the upper stem and bulge region of TAR, a structured element in the nascent viral RNA, and controls a DRB-sensitive step early in RNAPII transcription elongation that results in hyperphosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II. In nuclear extracts, HIV-1 Tat associates tightly with the CDK9-containing positive transcription elongation factor complex, PTEFb (4 -6). Recent studies indicate that Tat binds directly through its trans-activation domain to the cyclin subunit (CycT1) of the P-TEFb complex and induces loop sequencespecific binding of the P-TEFb complex to TAR RNA (7-9). Neither CycT1 nor the P-TEFb complex bind TAR RNA in the absence of Tat, and thus the binding is highly cooperative for both Tat and P-TEFb (7, 9). The Tat-CycT1 interaction requires zinc as well as cysteine residues in each protein and therefore may represent a metal-linked heterodimer (8). Tat appears to contact residues in the carboxyl-terminal boundary of the CycT1 cyclin domain which are not critical for binding of cyclin T1 to CDK9 (8, 10 -14), and basic residues in CycT1 (Arg-251 and Arg-254) further stabilize the Tat⅐P-TEFb⅐TAR RNA complex (8). Thus the assembly of this complex appears to involve a series of adaptive interactions between the trans-activation and arginine-rich motif (RNA binding) domains of Tat and their respective protein (CycT1) and nucleic acid (TAR) partners during transcription.These studies have raised the possibility that at least two separate events may govern the assembly of functional P-TEFb⅐Tat⅐TAR complexes. 1) The interaction of Tat with CycT1 induces a conformational change in Tat that enhances its affinity and kinetic stability for TAR RNA. 2) TAR RNA may enhance the affinity between CycT1 and Tat, through an "induced fit" mechanism. These two events would not necessarily be mutually exclusive, and both could contribute significantly to the assembly of a stable ternary complex necessary to position...

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Small unnatural peptides that target specific RNA structures have the potential to control biological processes. RNA−protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of human immunodeficiency virus type 1 (HIV-1) gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem−loop structure located at the 5‘-end of all nascent HIV-1 transcripts. We report here a synthetic peptide derived from Tat sequence (37−72), containing all d-amino acids, that binds in the major groove of TAR RNA and interferes with transcriptional activation by Tat protein in vitro and in HeLa cells. Our results indicate that unnatural peptides can inhibit the transcription of specific genes regulated by RNA−protein interactions.