P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo.
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.
Ni
Zhuoyu
Z
Department of Molecular Biology and Genetics,1 School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA.
Saunders
Abbie
A
Fuda
Nicholas J
NJ
Yao
Jie
J
Suarez
Jose-Ramon
JR
Webb
Watt W
WW
Lis
John T
JT
D000818:Animals; D029721:Drosophila Proteins / Q000502:physiology*; D004331:Drosophila melanogaster / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D042925:Positive Transcriptional Elongation Factor B / Q000502:physiology*; D012319:RNA Polymerase II / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic
D029721:Drosophila Proteins; D018840:HSP70 Heat-Shock Proteins; D014157:Transcription Factors; D042925:Positive Transcriptional Elongation Factor B; D012319:RNA Polymerase II