| HIV Tat protein is a small viral-encoded transcription factor under intense scrutiny as a potential drug target. Preferred high-resolution methods such as X-ray co-crystal diffraction and NMR solution structure determination are unavailable for mapping the interactions of Tat protein with its RNA ligand, TAR, due to limitations in solubility of the complex. In lieu of these biophysical methods, a high-resolution biochemical method was employed. Two novel iron-chelating amino acids, Nalpha-Fmoc-N epsilon-[EDTA(OEt)3]-L-lysine and N alpha-Fmoc-Nepsilon-[DTPA]-L-lysine, were synthesized as metal chelators for direct incorporation into Tat peptides during solid phase synthesis. These peptides bind TAR RNA with high affinity. The tethered iron generated diffusible hydroxyl radicals, which cut proximal nucleotides. The position of the cleaved nucleotides identified the location of the iron-bearing amino acid within the native complex. Position 45 in these Tat peptides was mapped on TAR RNA. Specificity for the wild-type RNA was dependant on the length of the peptide.; All of the peptides caused strong cleavage near the 3-nucleotide bulge, which is the binding site on TAR for Tat. However, the smaller peptides also cleaved sites at the two single-nucleotide bulges in the TAR stem, indicating that these peptides do not discriminate well between the cognate and nonspecific sites. Thus, the cleavage experiments with these engineered peptides demonstrated their usefulness for studying protein-nucleic acid interactions. |
