Tackling Tat

Activation of cellular genes typically involves control of transcription initiation by DNA-binding regulatory proteins. The human immunodeficiency virus transactivator protein, Tat, provides the first example of the regulation of viral gene expression through control of elongation by RNA polymerase II. In the absence of Tat, initiation from the long terminal repeat is efficient, but transcription is impaired because the promoter engages poorly processive polymerases that disengage from the DNA template prematurely. Activation of transcriptional elongation occurs following the recruitment of Tat to the transcription machinery via a specific interaction with an RNA regulatory element called TAR, a 59-residue RNA leader sequence that folds into a specific stem-loop structure. After binding to TAR RNA, Tat stimulates a specific protein kinase called TAK (Tat-associated kinase). This results in hyperphosphorylation of the large subunit of the RNA polymerase II carboxyl- terminal domain. The kinase subunit of TAK, CDK9, is analogous to a component of a positive acting elongation factor isolated from Drosophila called pTEFb. Direct evidence for the role of TAK in transcriptional regulation of the HIV long terminal repeat comes from experiments using inactive mutants of the CDK9 kinase expressed in trans to inhibit transcription. A critical role for TAK in HIV transcription is also demonstrated by selective inhibition of Tat activity by low molecular mass kinase inhibitors. A second link between TAK and transactivation is the observation that the cyclin component of TAK, cyclin T1, also participates in TAR RNA recognition. It has been known for several years that mutations in the apical loop region of TAR RNA abolish Tat activity, yet this region of TAR is not required for binding by recombinant Tat protein in vitro, suggesting that the loop region acts as a binding site for essential cellular co-factors. Tat is able to form a ternary complex with TAR RNA and cyclin T1 only when a functional loop sequence is present on TAR.