Tat is essential for viral replication and functions primarily by enhancing transcription elongation from the HIV promoter [68-72]. At the start of this project. Tat was known to recruit the heterodimeric kinase, P-TEFb, composed of CycTI (cyclin Tl) and Cdk9 subunits, to the TAR RNA element at the 5' end of nascent HIV transcripts, triggering RNA Pol II CTD phosphorylation and transcriptional elongation [70, 73, 74]. The recent crystal structure of a Tat-P-TEFb complex [75] was an important step forward, showing how both the CycTI and Cdk9 partners are required to template Tat structure. An immediate challenge is to define how the Tat-PTEFb complex regulates transcription. Two HARC Center discoveries changed the basic P-TEFb paradigm and provided new hypotheses that we will test. First using our proteomics pipeline, we found that Tat selectively recruits P-TEFb as part of a large, stoichiometric complex containing additional transcription elongation factors [3, 76] This complex has been called the super elongation complex (SEC) [77] and the MLL-fusion complex to reflect its role in promoting certain leukemias [78]. Here we refer to it as AFF4c to reflect the central scaffolding role of the AFF4 protein. Remarkably, AFF4c is not only part of a combinatorial family of related assemblies (AFFcs), but it also is normally recruited to only a subset of human genes occupied by paused polymerases [77-80]. This raises the question of the origin of the specificity of AFF4 assembly and recruitment which we aim to address. Second, we found that Tat assembles at the HIV promoter along with an inactive, 7SK snRNP-associated form of PTEFb [70] which is dismantled upon TAR binding [4]. By dissecting the host complexes recruited by Tat we will fill major gaps in understanding HIV and host transcription. This mechanistic insight is needed to target Tat with therapeutics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-AARR-K)
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University of California San Francisco
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