P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo

Mol Cell Biol. 2008 Feb;28(3):1161-70. doi: 10.1128/MCB.01859-07. Epub 2007 Dec 10.

Abstract

Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Drosophila Proteins / physiology*
  • Drosophila melanogaster / genetics
  • HSP70 Heat-Shock Proteins / genetics
  • Positive Transcriptional Elongation Factor B / physiology*
  • RNA Polymerase II / metabolism*
  • Transcription Factors
  • Transcription, Genetic*

Substances

  • Drosophila Proteins
  • HSP70 Heat-Shock Proteins
  • Transcription Factors
  • Positive Transcriptional Elongation Factor B
  • RNA Polymerase II