HIV encodes a novel regulatory protein, (Tat), which is important for virus growth, pathogenesis and latency. It encodes a transacting stimulator of HIV gene expression. A cis acting element called TAR, located in the long terminal repeat (LTR), downstream of the transcription initiation site in the provirus, is required for transactivation by Tat. The mechanism whereby Tat stimulates HIV gene expression remains uncertain. It is generally accepted that its major action is at the level of transcription, although the extent to which the stimulation is due to effects on transcriptional initiation or elongation is still unresolved. However, it is well-established that Tat can stimulate at the elongation level, both in vivo and in vitro. The present proposal is based on the hypothesis that transcription complexes formed at the HIV promoter in the absence of Tat are unstable during elongation and that TAR RNA forms a scaffold for the binding of Tat and of cellular proteins which alter the complexes so as to increase their elongational competence. The principal objective of the proposed collaboration is to explore the mechanism underlying this process and to discover the identity of the transcription factor(s) that are recruited into the complex by Tat and TAR. To this end, the project will exploit in vitro transcription systems of three types; (i) a conventional HeLa cell nuclear extract that responds to Tat, (ii) a system in which the DNA template is immobilized on agarose beads so that components can be added and removed at will, and (iii) a system directed by templates carrying heterologous attenuator signals that can cause polymerase pausing and termination.
