Open AccessP-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo
Author(s) -
Zhuoyu Ni,
Abbie Saunders,
Nicholas J. Fuda,
Jie Yao,
José-Ramón Suárez,
Watt W. Webb,
John T. Lis
Publication year - 2008
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.01859-07
Subject(s) - p tefb , rna polymerase ii , biology , microbiology and biotechnology , elongation factor , transcription (linguistics) , polymerase , transcription factor ii d , rna polymerase , rna , gene expression , gene , genetics , promoter , ribosome , linguistics , philosophy
P ositivet ranscriptione longationf actor 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 activeDrosophila 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 theHsp70 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 theHsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.