NELF, a Multisubunit Complex Containing RD, Cooperates with DSIF to Repress RNA Polymerase II Elongation

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Abstract

DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.

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Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase.

N. Marshall, J. Peng, Z. Xie … (1996)

The entry of RNA polymerase II into a productive mode of elongation is controlled, in part, by the postinitiation activity of positive transcription elongation factor b (P-TEFb) (Marshall, N. F., and Price, D. H. (1995) J. Biol. Chem. 270, 12335-12338). We report here that removal of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II abolishes productive elongation. Correspondingly, we found that P-TEFb can phosphorylate the CTD of pure RNA polymerase II. Furthermore, P-TEFb can phosphorylate the CTD of RNA polymerase II when the polymerase is in an early elongation complex. Both the function and kinase activity of P-TEFb are blocked by the drugs 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and H-8. P-TEFb is distinct from transcription factor IIH (TFIIH) because the two factors have no subunits in common, P-TEFb is more sensitive to DRB than is TFIIH, and most importantly, TFIIH cannot substitute functionally for P-TEFb. We propose that phosphorylation of the CTD by P-TEFb controls the transition from abortive into productive elongation mode.

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

D. Price, Kristin N. Marshall (1995)

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.