Mapping of HIV-1 Tat protein sequences required for binding to Tar RNA

We have utilized a gel retardation assay to study the binding of chemically synthesized domains of the HIV-1 Tat protein to radiolabeled trans-activating response element (Tar) RNA. As with recombinant Tat protein, synthetic Tat specifically binds to Tar RNA and not to a defective Tar RNA or to anti-sense Tar RNA. The 6 amino acid portion of the basic region containing five arginines is sufficient to confer Tar binding to overlapping Tat protein fragments; Tat fragments that lack the basic region do not bind Tar. In addition, the basic region alone can also bind Tar RNA; however, binding of the basic region is non specific since defective Tar RNA is bound as well as wild type Tar RNA. Binding specificity for wild type Tar RNA can be conferred by the addition of a minimum of 8 random amino acids to either end of the basic region.