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Molecular and Cellular Biology, February 2008, p. 1161-1170, Vol. 28, No. 3
0270-7306/08/$08.00+0     doi:10.1128/MCB.01859-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

P-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo{triangledown}

Zhuoyu Ni,1 Abbie Saunders,1 Nicholas J. Fuda,1 Jie Yao,2 Jose-Ramon Suarez,3 Watt W. Webb,2 and John T. Lis1*

Department of Molecular Biology and Genetics,1 School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853,2 Aventis Pharmaceuticals Inc., Bridgewater, New Jersey 088073

Received 11 October 2007/ Returned for modification 31 October 2007/ Accepted 15 November 2007

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.

* Corresponding author. Mailing address: 416 Biotechnology Building, Cornell University, Ithaca, NY 14850. Phone: (607) 255-2442. Fax: (607) 255-2428. E-mail: jtl10{at}cornell.edu

{triangledown} Published ahead of print on 10 December 2007.

Molecular and Cellular Biology, February 2008, p. 1161-1170, Vol. 28, No. 3
0270-7306/08/$08.00+0     doi:10.1128/MCB.01859-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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