ORIENTATION-SPECIFIC CIS COMPLEMENTATION BY BULGE-MUTATED AND LOOP-MUTATED HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAR RNAS

Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA. TAR comprises a partially base paired stem and hexanucleo tide loop with a tripyrimidine bulge in the upper stem. In vitro, Tat binds to the bulge and upper stem, with no requirement For the loop. H owever, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway. Given that activation appears to be a two-component system co mprising a Tat-bulge interaction and a cellular factor-loop interactio n, we considered that it might be possible to spatially separate the t wo components and retain activation. We have constructed a series of d ouble TAR elements comprising various combinations of mutated TAR stru ctures. Defective TARs with nucleotide substitutions in either the bul ge or the loop complemented each other to give wild-type activation. H owever, the complementation was orientation specific, requiring the in tact Tat binding site to reside on the 5'-proximal TAR. These data sug gest that provided the wild-type orientation of the bulge and loop ele ment is retained, there is no requirement for them to coexist on the s ame TAR structure.