A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS

Abstract

Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-␤-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF͞ NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF͞NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF͞NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires >18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of backtracked nascent RNA, TFIIS inhibition may help DSIF͞NELF negatively regulate productive transcription.transcription elongation ͉ pausing ͉ backtracking R egulation of productive mRNA chain synthesis by RNA polymerase II (RNAPII) occurs when the RNA chain is Ϸ10-100 nt long and determines whether RNAPII forms a fully functional transcription elongation complex (TEC) or halts RNA synthesis during the early stages of transcript elongation (reviewed in refs. 1-5). Conversion to a productive TEC involves an ordered set of transitions that require successive phosphorylation of Ser-5 and Ser-2 in the RPB1 C-terminal heptapeptide repeat domain (CTD) by kinase components of TFIIH and positive elongation factor P-TEFb, respectively. These CTD changes orchestrate release of initiation factors, recruitment of general elongation and chromatin-modifying factors, and capping of the nascent transcript by the capping enzyme. Completion of these steps is required to produce a TEC able to transcribe through nucleosomes on a chromatin template and synthesize full-length pre-mRNAs.

Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex

“…4C). These results are consistent with the finding that human DSIF and NELF require transcripts ≥18 nt long to inhibit transcription (31), and also a recent study showed human DSIF preferentially bound elongation complexes containing transcripts that were at least 25 nt long (32). The 5′ end of an 18-nt-long nascent transcript just begins to emerge from the surface of Pol II (28,33).…”

Regulation of clustered gene expression by cofactor of BRCA1 (COBRA1) in breast cancer cells

“…In vitro biochemical characterization clearly demonstrate that NELF, of which COBRA1 is an integral subunit, modulates transcription elongation by stalling RNAPII at the promoter-proximal region (Yamaguchi et al, 1999;Palangat et al, 2005). Previous in vivo studies in human and Drosophila systems corroborate the RNAPII-stalling function of NELF in gene regulation (Wu et al, , 2005aAiyar et al, 2004).…”

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

“…Thus the MED31/SOH1 subunit of the Mediator displays genetic interactions not only with Pol II (34), but also with TFIIB (34) and several transcription elongation factors that include SII (35), the last in turn displaying close relationships with hSpt4-hSpt5/DSIF (36)(37)(38)(39). Furthermore, consistent with its role in early elongation, TFIIB has also been connected to diverse elongation and processing factors (40).…”