Isolation and functional characterization of P-TEFb-associated factors that control general and HIV-1 transcriptional elongation.
Originally identified as a factor crucial for RNA polymerase (Pol) II 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.
Chen
Ruichuan
R
Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian, China.
Liu
Min
M
Zhang
Kai
K
Zhou
Qiang
Q
D017930:Genes, Reporter; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D042925:Positive Transcriptional Elongation Factor B / Q000737:chemistry / Q000302:isolation & purification*; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification*; D014158:Transcription, Genetic
D014157:Transcription Factors; D042925:Positive Transcriptional Elongation Factor B
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