High Affinity Binding of TAR RNA by the Human Immunodeficiency Virus Type-1 tat Protein Requires Base-pairs in the RNA Stem and Amino Acid Residues Flanking the Basic Region

Abstract

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). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1·07±0·05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26·C39 and A27·U38 reduced tat affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22·U40 to U·A reduced tat affinity 5-fold, and mutation of G21·C41 to C·G reduced tat affinity 4-fold.The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23→C mutation in the bulge, or G26·C39→C·G mutation in the stem of TAR RNA. By contrast, the ADP-1 (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23→C or G26·C39→C·G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively.Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compared the binding properties of the tat protein and the tat-derived peptides. Binding of the tat protein and the ADP-1 peptide is strongly inhibited by modification of the purines flanking the bulge, G21, A22, G26 and A27. This suggests that both the protein and the peptide make contact with bases in the distorted major groove of TAR near the bulge. The ethylation interference experiments show that the binding of tat protein is also inhibited by modification of the P22 phosphate near the bulge. Binding of both the tat protein and the ADP-1 peptide, is also strongly inhibited by modification of phosphates located on the strand opposite the bulge (P36 to P40). By contrast, modification of the P36 to P40 phosphates is much less disruptive of the binding of the ADP-3 and ADP-5 peptides.We conclude that tat forms multiple contacts with TAR RNA involving U23, specific base-pairs in the stem, and phosphates on both strands of the TAR RNA near the bulge. Peptides carrying only the basic region and C-terminal extensions appear to bind TAR RNA not only more weekly but also relatively non-specifically, whereas peptides that contain flanking residues from the "core" region of the protein are able to recognize TAR RNA with a specificity that closely resembles that of the tat protein.