HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TRANSACTIVATOR PROTEIN, TAT, STIMULATES TRANSCRIPTIONAL READ-THROUGH OF DISTAL TERMINATOR SEQUENCES IN-VITRO

The human immunodeficiency virus type 1 transactivator protein, tat, s pecifically stimulates transcription from the viral long terminal repe at. We used cell-free transcription systems to test whether tat can st imulate transcriptional read-through of an artificial terminator seque nce (e.g., a stable RNA stem-loop structure followed by a tract of nin e uridine residues) placed downstream of the viral long terminal repea t. In the absence of tat, RNA polymerases are prematurely released fro m the template at the terminator sequence. Recombinant tat protein pur ified from Escherichia coli increased the synthesis of full-length tra nscripts almost-equal-to 25-fold and decreased the amount of transcrip ts ending at the terminator sequence. The reaction is strictly depende nt upon the presence of a functional transactivation-responsive region (TAR) sequence. Mutations in the tat binding site on TAR RNA and muta tions in the TAR RNA loop block transactivation in vivo. Neither type of mutation is able to respond to tat in vitro. These results strongly suggest that after transcription through the TAR region, tat modifies the transcription complex to increase its elongation capacity.