Purification of P-TEFb, a transcription factor required for the transition into productive elongation

Abstract

Production of full-length runoff transcripts in vitro and functional mRNA in vivo is sensitive to the drug 5,6-dichloro-1-beta-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 Kc 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 run-off transcripts.