Structure-based design of a dimeric RNA-peptide complex

The arginine-rich RNA-binding domain of bovine immunodeficiency virus (BIV) Tat adopts a beta -hairpin conformation upon binding to the major groove o f BIV TAR. Based on its NMR structure, we modeled dimeric arrangements in w hich two adjacent TAR sites might be recognized with high affinity by a dim eric peptide. Some dimeric RNAs efficiently bound two unlinked BIV Tat pept ides in vitro, but could not bind even one monomeric peptide in vivo, as mo nitored by transcriptional activation of human immunodeficiency virus long terminal repeat reporters. Results with additional reporters suggest that e xtending the RNA helix in the dimeric arrangements inhibits peptide binding by decreasing major groove accessibility, In contrast, a dimeric peptide e fficiently bound an optimally arranged dimeric TAR in vivo, and bound with an affinity at least 10-fold higher than the monomeric peptide in vitro. Mu tating specific nucleotides in each RNA 'half-site' or specific amino acids in each beta -hairpin of the dimeric peptide substantially decreased bindi ng affinity, providing evidence for the modeled dimer-dimer interaction. Th ese studies provide a starting point for identifying dimeric RNA-protein in teractions with even higher binding affinities and specificities.