HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT IS AN INTEGRAL COMPONENT OF THE ACTIVATED TRANSCRIPTION-ELONGATION COMPLEX

The human immunodeficiency virus type 1 transactivator protein, Tat, s timulates transcriptional elongation from the viral long terminal repe at, To test whether Tat associates directly with activated transcripti on complexes, we have used the lac repressor protein (LacR) to ''trap' ' elongating RNA polymerases. The arrested transcription complexes wer e purified by binding biotinylated templates to streptavidin-coated ma gnetic beads. Transcription complexes were released from the magnetic beads following cleavage of the templates with restriction enzymes and were immunoblotted with antibodies to Tat, LacR and RNA polymerase II , The Tat protein copurified with RNA polymerase bound to wild-type te mplates but did not copurify with transcription complexes prepared by using templates carrying mutations in the transactivation response ele ment (TAR) RNA, We conclude that Tat and cellular cofactors become att ached to the transcription complex during its transit through TAR.