Isolation and functional characterization of P-TEFb-associated factors that control general and HIV-1 transcriptional elongation

Abstract

Originally identified as a factor crucial for RNA polymerase (Pol)11 transcriptional elongation of cellular genes, the P-TEFb kinase was subsequently shown to also serve as a specific host co-factor required for HIV-1 transcription. Recruited by either the bromodomain protein Brd4 to cellular promoters for general transcription or the HIV-1 Tat protein to the viral LTR for activated HIV-1 transcription, P-TEFb stimulates the processivity of Pol II through phosphorylating the C-terminal domain of Pol II and a pair of negative elongation factors, leading to the synthesis of full-length transcripts. However, abundant evidence indicates that P-TEFb does not act alone in the cell and that all of its known biological functions are likely mediated through the interactions with various regulators. Although a number of P-TEFb-associated factors have already been identified, there are likely more yet to be discovered. Given that P-TEFb plays an essential role in HIV-1 transcription, a major challenge facing the field is to identify all the P-TEFb-associated factors and determine how they may modulate Tat-transactivation and HIV-1 replication. Described here is a set of experimental procedures that have not only enabled us to isolate and identify several P-TEFb-associated factors, but also provided the means to characterize their biochemical functions in HIV-1 transcriptional control. In light of the recent demonstrations that transcriptional elongation plays a much more important role in controlling metazoan gene expression than previously thought, the techniques presented here will also be useful for analyzing Pol II elongation of cellular genes. (C) 2010 Elsevier Inc. All rights reserved.