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

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.

ACKNOWLEDGMENTS

This work was supported by NIH grant GM25232 to J.T.L. and NSF grant CHE-0242328 to W.W.W. and J.T.L.

We thank D. H. Price for anti-cyclin T serum and Lis lab members for comments on the manuscript.

Z.N. and J.T.L. conceptualized the project; A.S. and J.T.L. wrote the manuscript; Z.N. obtained the data in Fig. 1 and 2 and purified recombinant CstF50 for antiserum production; A.S. obtained the data in Fig. 3; N.J.F. obtained the data in Fig. 4; J.Y. obtained the data in Fig. 5; J.-R.S. provided the FP; W.W.W. advised and provided support for the work presented in Fig. 5.