Previous mutagenic analysis of TAR indicates that the bulge residues and a 4 bp segment of the stem constitute, in part, the tat binding site. However, there appears to be no sequence-specific contribution of the six-base loop. We have employed a ribonuclease protection technique to explore the interaction of tat with single-stranded regions of TAR. The results indicate that tat interacts with both the bulge and loop regions of TAR.
The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.
The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA(K1) was competed against 3H-labelled mutant TAR RNA(K2).
Efficient transcription from the human immunodeficiency virus(HIV) promoter depends on binding of the viral regulatory protein Tat to a cis-acting RNA regulatory element, TAR. Tat binds at a trinucleotide bulge located near the apex of the TAR stem-loop structure.
The Human Immunodeficiency Virus type 1 Tat protein interacts specifically with a U-rich bulge within an RNA stem-loop known as the trans-activation responsive region(TAR) that occurs in all viral transcripts. We have identified the cross-linked amino acid as Arg55 in the basic region of the Tat peptide by use of a combination of proteolytic digestions and MALDI-TOF mass spectrometric analysis.
Short basic peptides that competed full-length tat from complexes with TAR RNA also inhibited tat stimulation of transcription, whereas short basic peptide unable to bind TAR or compete tat from complexes were also unable to inhibit tat stimulation of transcription. These data confirm that active HIV-1 tat must first interact with TAR RNA via basic amino acid residues in order to stimulate transcription of downstream sequences.
Electron paramagnetic resonance (EPR) spectroscopy was utilized to investigate the correlation between RNA structure and RNA internal dynamics in complexes of HIV-1 TAR RNA with small molecules. TAR RNAs containing single nitroxide spin-labels in the 2'-position of U23, U25, U38, or U40 were incubated with compounds known to inhibit TAR-Tat complex formation.
HIV-1 TAR element is the target of the viral transactivating Tat protein, which is expressed during the course of HIV-1 infection and known to act at the RNA level to enhance viral gene expression by >100-fold (29). We thus examined whether Tat could shield TAR RNA and prevent its processing by Dicer.
