title: The RPB2 flap loop of human RNA polymerase II is dispensable for transcription initiation and elongation.
abstract: The flap domain of multisubunit RNA polymerases (RNAPs), also called the wall, forms one side of the RNA exit channel. In bacterial RNAP, the mobile part of the flap is called the flap tip and makes essential contacts with initiation and elongation factors. Cocrystal structures suggest that the orthologous part of eukaryotic RNAPII, called the flap loop, contacts transcription factor IIB (TFIIB), but the function of the flap loop has not been assessed. We constructed and tested a deletion of the flap loop in human RNAPII (subunit RPB2 Δ873-884) that removes the flap loop interaction interface with TFIIB. Genome-wide analysis of the distribution of the RNAPII with the flap loop deletion expressed in a human embryonic kidney cell line (HEK 293) revealed no effect of the flap loop on global transcription initiation, RNAPII occupancy within genes, or the efficiency of promoter escape and productive elongation. In vitro, the flap loop deletion had no effect on promoter binding, abortive initiation or promoter escape, TFIIS-stimulated transcript cleavage, or inhibition of transcript elongation by the complex of negative elongation factor (NELF) and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). A modest effect on transcript elongation and pausing was suppressed by TFIIF. Although similar to the flap tip of bacterial RNAP, the RNAPII flap loop is not equivalently essential.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Murali
authors.initials: M
authors.lastname: Palangat
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Grass
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Langelier
authors.forename: Benoit
authors.initials: B
authors.lastname: Coulombe
authors.forename: Robert
authors.initials: R
authors.lastname: Landick
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D006801:Humans; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics*; D017384:Sequence Deletion; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
chemical_list: D009687:Nuclear Proteins; C112224:SUPT5H protein, human; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D035602:Transcriptional Elongation Factors; C118437:negative elongation factor; D012319:RNA Polymerase II; C056604:transcription factor TFIIF