THE HUMAN-IMMUNODEFICIENCY-VIRUS LONG TERMINAL REPEAT INCLUDES A SPECIALIZED INITIATOR ELEMENT WHICH IS REQUIRED FOR TAT-RESPONSIVE TRANSCRIPTION

The effects of mutations in human immunodeficiency virus type-1 (HIV-1 ) long terminal repeat on initiation and on Tat-mediated trans-activat ion were studied using cell-free transcription assays. All the element s that are necessary for efficient transcription initiation in vitro a re included in the core promoter. This region contains three tandem Sp 1 binding sites, a TATA element and an initiator (INR) sequence. Altho ugh the HIV-1 INR element overlaps the trans-activation response regio n (TAR), it forms an integral part of the promoter. The HIV-1 INR elem ent was characterised in detail using a template that carries a comple te HIV-1 promoter and a displaced TAR RNA element. The results demonst rate that the sequence G(+1)GGTCT is essential for HIV-1 INR function. RNase protection experiments show that Tat acts exclusively to stimul ate transcriptional elongation. Mutations in the core promoter element s reduce initiation rates dramatically but do not block Tat activity. For each mutation studied, the total level of transcription in the pre sence of Tat is proportional to the rate of initiation in the absence of Tat. Furthermore the rate of initiation remains constant in the pre sence or absence of Tat. We conclude that the elements of the HIV-1 co re promoter act in concert to simulate initiation. By contrast, Tar ac ts independently of the core promoter elements and stimulates elongati on. The data strongly suggest that Tat is recruited to the elongating transcription complex during its transit through TAR.