Detailed mutational analysis of TAR RNA: critical spacing between the bulge and loop recognition domains.

Trans-activation of HIV-1 by the Tat protein is mediated through a cis-acting element (TAR) in the viral RNA. In order to obtain further insight into the molecular interactions for trans-activation, a detailed mutational analysis of TAR RNA was carried out. TAR RNA forms a hairpin structure with important sequence elements in the single-stranded bulge- and loop-domains. We found that the sequence of the base-pairs flanking the bulge is critical for Tat-mediated trans-activation. In addition, Tat-response is reduced when the bulge is forced into a base-paired configuration through the introduction of complementary nucleotides on the opposite side of the stem. Thus, the 3-nucleotide bulge and adjacent base-pairs comprise a recognition domain with both sequence- and structure-elements. Accessibility of the loop sequences is also important for Tat function, since base-pairing through the formation of a pseudoknot-like structure does inhibit Tat action. A third critical parameter that influences the magnitude of Tat response is the number of loop nucleotides. Finally, the relative spacing between the loop and the bulge is also important. We introduced additional base-pairs in the stem connecting the two domains. Such mutations progressively decreased the efficiency of Tat induction. Interestingly, activity of the HIV-2 Tat protein did markedly increase on targets with one or two additional basepairs. These results suggest that Tat interacts with a cellular loop-binding protein(s) to increase HIV gene expression.