THE RNA ELEMENT ENCODED BY THE TRANS-ACTIVATION-RESPONSIVE REGION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 IS FUNCTIONAL WHEN DISPLACED DOWNSTREAM OF THE START OF TRANSCRIPTION

The human immunodeficiency virus type 1 (HIV-1) trans-activator protei n, Tat, specifically stimulates transcription from the viral long term inal repeat. Tat binds to an RNA stem-loop structure encoded by the tr ans-activation response region (TAR). To test whether TAR is functiona l when displaced downstream of the start of transcription, we assayed a series of templates carrying duplicated TAR elements in cell-free tr anscription systems. When the normally positioned TAR element (TAR-1) is inactivated by mutations in either the Tat binding site or the apic al loop sequence, which acts as the binding site for a cellular factor , trans-activation can be rescued by a wild-type TAR element placed do wnstream (TAR-2). The TAR-2 element is functional even when placed >20 0 nt downstream of TAR-1. TAR complementation experiments have also sh own that a functional TAR element requires both an intact Tat binding site and an intact apical loop sequence. For example, if TAR-1 carries a mutation in the loop element it cannot be rescued by a TAR-2 elemen t carrying a mutation in the Tat binding site. Substitution mutations in TAR-1 show that the 5' half of TAR also encodes an essential DNA el ement which is required for efficient transcription initiation. These results strongly suggest that Tat and cellular cofactors which bind TA R RNA associate with the transcription complex during its transit thro ugh TAR.