Purification of P-TEFb, a transcription factor required for the transition into productive elongation.
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.
Marshall
N F
NF
Department of Biochemistry, University of Iowa, Iowa City 52242, USA.
Price
D H
DH
D000818:Animals; D002474:Cell-Free System; D002478:Cells, Cultured; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology*; D004331:Drosophila melanogaster / Q000166:cytology; D005786:Gene Expression Regulation; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis*; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D012333:RNA, Messenger; D014157:Transcription Factors; D004004:Dichlororibofuranosylbenzimidazole; D012319:RNA Polymerase II