Purification of P-TEFb, a Transcription Factor Required for the Transition into Productive Elongation

doi:10.1074/jbc.270.21.12335

Purification of P-TEFb, a Transcription Factor Required for the Transition into Productive Elongation (*)

Nick F. Marshall and
David H. Price(§)

From the (1)Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242

^§ To whom correspondence should be addressed. Tel.: 319-335-7910; Fax: 319-335-9570.

Abstract

Production of full-length runoff transcripts in vitro and functional mRNA in vivo is sensitive to the drug 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). We previously proposed the existence of an activity, P-TEF (positive transcription elongation factor) that functions in a DRB-sensitive manner to allow RNA polymerase II elongation complexes to efficiently synthesize long transcripts (Marshall, N. F. and Price, D. H.(1992) Mol. Cell. Biol. 12, 2078-2090). We have fractionated nuclear extracts of Drosophila melanogaster K^c cells and identified three activities, P-TEFa, factor 2, and P-TEFb, that are directly involved in reconstructing DRB-sensitive transcription. P-TEFb is essential for the production of DRB-sensitive long transcripts in vitro, while P-TEFa and factor 2 are stimulatory. P-TEFb activity is associated with a protein comprising two polypeptide subunits with apparent molecular masses of 124 and 43 kDa. Using a P-TEFb-dependent transcription system, we show that P-TEFb acts after initiation and is the limiting factor in the production of long runoff transcripts.

Footnotes

↵* This work was supported by National Institutes of Health Grant R01-GM35500. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

HIV

human immunodeficiency virus

DRB

5,6-dichloro-1-β-D-ribofuranosylbenzimidazole

P-TEF and N-TEF

positive and negative transcription elongation factors, respectively

FT

flow-through fraction.
↵² Z. Xie and D. H. Price, unpublished data.
↵³ L. Zawel, D. Reinberg, N. F. Marshall, and D. H. Price, unpublished data.
↵⁴ D. R. Chafin and D. H. Price, unpublished data.
↵⁵ M. Doro and D. H. Price, unpublished data.