TRANSFER OF TAT AND RELEASE OF TAR RNA DURING THE ACTIVATION OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TRANSCRIPTION ELONGATION COMPLEX

The HIV-1 trans-activator protein, Tat, is a potent activator of trans criptional elongation, Tat is recruited to the elongating RNA polymera se during its transit through the trans-activation response region (TA R) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain, We have shown that transcription complexes that ha ve acquired Tat produce 3-fold more full-length transcripts than compl exes not exposed to Tat. Western blotting experiments demonstrated tha t Tat is tightly associated with the paused polymerases, To determine whether TAR RNA also becomes attached to the transcription complex, DN A oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H, After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcri ption complex. Even after the removal of TAR RNA, transcription comple xes that have been activated by Tat show enhanced processivity: We con clude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RN A does not play a direct role in the activation of elongation and is u sed simply to recruit Tat and cellular co-factors.