Biology:P-TEFb

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The positive transcription elongation factor, P-TEFb, is a multiprotein complex that plays an essential role in the regulation of transcription by RNA polymerase II (Pol II) in eukaryotes.[1] Immediately following initiation Pol II becomes trapped in promoter proximal paused positions on the majority of human genes (Figure 1).[2][3] P-TEFb is a cyclin dependent kinase that can phosphorylate the DRB sensitivity inducing factor (DSIF)[4] and negative elongation factor (NELF),[5] as well as the carboxyl terminal domain of the large subunit of Pol II[6] and this causes the transition into productive elongation leading to the synthesis of mRNAs. P-TEFb is regulated in part by a reversible association with the 7SK snRNP.[7] Treatment of cells with the P-TEFb inhibitors DRB or flavopidirol leads to loss of mRNA production and ultimately cell death.[6][8]

Discovery

P-TEFb was identified and purified as a factor needed for the generation of long run-off transcripts using an in vitro transcription system derived from Drosophila cells.[9]

Components

It is a cyclin dependent kinase containing the catalytic subunit, Cdk9, and a regulatory subunit, cyclin T in Drosophila.[10] In humans there are multiple forms of P-TEFb which contain Cdk9 and one of several cyclin subunits, cyclin T1, T2, and K.[11][12] P-TEFb associates with other factors including the bromodomain protein BRD4,[13] and is found associated with a large complex of proteins called the super elongation complex.[14][15] Importantly, for the AIDS virus, HIV, P-TEFb is targeted by the HIV Tat protein[16] which bypasses normal cellular P-TEFb control and directly brings P-TEFb to the promoter proximal paused polymerase in the HIV genome.[17][18]

Structure

Figure 2. Structure of P-TEFb bound by HIV Tat. PDB ID: 3MIA Cdk9 (blue), cyclin T1 (cyan), Tat (orange), ATP (magenta), magnesium (purple), zinc atoms (yellow).

The structures of human P-TEFb containing Cdk9 and cyclin T1 and the HIV Tat•P-TEFb complex have been solved using X-ray crystallography. The first structure solved demonstrated that the two subunits were arranged as has been found in other cyclin dependent kinases.[19] Three amino acid substitutions were inadvertently introduced in the subunits used for the original structure and a subsequent structure determination using the correct sequences demonstrated the same overall structure except for a few significant changes around the active site.[20] The structure of HIV Tat bound to P-TEFb demonstrated that the viral protein forms extensive contacts with the cyclin T1 subunit (Figure 2).[20]

Regulation

Figure 3. Reversible association of P-TEFb with the 7SK snRNP. P-TEFb is released from the 7SK snRNP by Brd4 or HIV Tat. HEXIM is ejected and the two proteins are replaced by hnRNPs. The reverse of this process requires other unknown factors.

Because of its central role in controlling eukaryotic gene expression, P-TEFb is subject to stringent regulation at the level of transcription of the genes encoding the subunits, translation of the subunit mRNAs, turnover of the subunits, and also by an unusual mechanism involving the 7SK snRNP.[7] As shown in Figure 3 P-TEFb is held in the 7SK snRNP by the double stranded RNA binding protein HEXIM (HEXIM1 or HEXIM2 in humans). HEXIM bound to 7SK RNA or any double stranded RNA binds to P-TEFb and inhibits the kinase activity.[21][22] Two other proteins are always found associated with 7SK RNA. The methyl phosphase capping enzyme MEPCE puts a methyl group on the gamma phosphate of the first nucleotide of the 7SK RNA[23] and the La related protein LARP7 binds to the 3' end of 7SK.[24][25] When P-TEFb is extracted from the 7SK snRNP, 7SK RNA undergoes a conformation change, HEXIM is ejected and hnRNPs take the place of the factors removed.[7] The re-sequestration of P-TEFb requires another rearrangement of the RNA, binding of HEXIM and then P-TEFb. In rapidly growing cells the 7SK snRNP is the predominant form of P-TEFb. For review.[26]

References

  1. ↑ "RNA polymerase II elongation control". Annual Review of Biochemistry 81: 119–43. 2012. doi:10.1146/annurev-biochem-052610-095910. PMID 22404626. 
  2. ↑ "c-Myc regulates transcriptional pause release". Cell 141 (3): 432–445. April 2010. doi:10.1016/j.cell.2010.03.030. PMID 20434984. 
  3. ↑ "Functional association of Gdown1 with RNA polymerase II poised on human genes". Molecular Cell 45 (1): 38–50. January 2012. doi:10.1016/j.molcel.2011.10.022. PMID 22244331. 
  4. ↑ "DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs". Genes & Development 12 (3): 343–356. February 1998. doi:10.1101/gad.12.3.343. PMID 9450929. 
  5. ↑ "NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation". Cell 97 (1): 41–51. April 1999. doi:10.1016/s0092-8674(00)80713-8. PMID 10199401. 
  6. ↑ 6.0 6.1 "Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase". The Journal of Biological Chemistry 271 (43): 27176–27183. October 1996. doi:10.1074/jbc.271.43.27176. PMID 8900211. 
  7. ↑ 7.0 7.1 7.2 "7SK snRNA: a noncoding RNA that plays a major role in regulating eukaryotic transcription". Wiley Interdisciplinary Reviews. RNA 3 (1): 92–103. 2012. doi:10.1002/wrna.106. PMID 21853533. 
  8. ↑ "Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo". The Journal of Biological Chemistry 276 (34): 31793–31799. August 2001. doi:10.1074/jbc.M102306200. PMID 11431468. 
  9. ↑ "Purification of P-TEFb, a transcription factor required for the transition into productive elongation". The Journal of Biological Chemistry 270 (21): 12335–12338. May 1995. doi:10.1074/jbc.270.21.12335. PMID 7759473. 
  10. ↑ "Identification of a cyclin subunit required for the function of Drosophila P-TEFb". The Journal of Biological Chemistry 273 (22): 13855–13860. May 1998. doi:10.1074/jbc.273.22.13855. PMID 9593731. 
  11. ↑ "Cyclin K functions as a CDK9 regulatory subunit and participates in RNA polymerase II transcription". The Journal of Biological Chemistry 274 (49): 34527–34530. December 1999. doi:10.1074/jbc.274.49.34527. PMID 10574912. 
  12. ↑ "Identification of multiple cyclin subunits of human P-TEFb". Genes & Development 12 (5): 755–762. March 1998. doi:10.1101/gad.12.5.755. PMID 9499409. 
  13. ↑ "Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4". Molecular Cell 19 (4): 535–545. August 2005. doi:10.1016/j.molcel.2005.06.029. PMID 16109377. 
  14. ↑ "The super elongation complex (SEC) and MLL in development and disease". Genes & Development 25 (7): 661–672. April 2011. doi:10.1101/gad.2015411. PMID 21460034. 
  15. ↑ "HIV-1 Tat and host AFF4 recruit two transcription elongation factors into a bifunctional complex for coordinated activation of HIV-1 transcription". Molecular Cell 38 (3): 428–438. May 2010. doi:10.1016/j.molcel.2010.04.013. PMID 20471948. 
  16. ↑ "Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product". Nature 330 (6147): 489–493. 1987. doi:10.1038/330489a0. PMID 2825027. 
  17. ↑ "Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro". Genes & Development 11 (20): 2622–2632. October 1997. doi:10.1101/gad.11.20.2622. PMID 9334325. 
  18. ↑ "HIV-1 Tat interacts with cyclin T1 to direct the P-TEFb CTD kinase complex to TAR RNA". Cold Spring Harbor Symposia on Quantitative Biology 63: 371–380. 1998. doi:10.1101/sqb.1998.63.371. PMID 10384302. 
  19. ↑ "The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation". The EMBO Journal 27 (13): 1907–1918. July 2008. doi:10.1038/emboj.2008.121. PMID 18566585. 
  20. ↑ 20.0 20.1 "Crystal structure of HIV-1 Tat complexed with human P-TEFb". Nature 465 (7299): 747–751. June 2010. doi:10.1038/nature09131. PMID 20535204. 
  21. ↑ "HEXIM1 is a promiscuous double-stranded RNA-binding protein and interacts with RNAs in addition to 7SK in cultured cells". Nucleic Acids Research 35 (8): 2503–2512. 2007. doi:10.1093/nar/gkm150. PMID 17395637. 
  22. ↑ "Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor". The EMBO Journal 23 (13): 2608–2619. July 2004. doi:10.1038/sj.emboj.7600275. PMID 15201869. 
  23. ↑ "Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme". Molecular Cell 27 (2): 262–274. July 2007. doi:10.1016/j.molcel.2007.06.027. PMID 17643375. 
  24. ↑ "LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated". Nucleic Acids Research 36 (7): 2219–2229. April 2008. doi:10.1093/nar/gkn061. PMID 18281698. 
  25. ↑ "A La-related protein modulates 7SK snRNP integrity to suppress P-TEFb-dependent transcriptional elongation and tumorigenesis". Molecular Cell 29 (5): 588–599. March 2008. doi:10.1016/j.molcel.2008.01.003. PMID 18249148. 
  26. ↑ "Cracking the control of RNA polymerase II elongation by 7SK snRNP and P-TEFb". Nucleic Acids Research 44 (16): 7527–7539. September 2016. doi:10.1093/nar/gkw585. PMID 27369380. 



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