title: Identification of Regions in the Spt5 Subunit of DRB Sensitivity-inducing Factor (DSIF) That Are Involved in Promoter-proximal Pausing.
abstract: DRB sensitivity-inducing factor (DSIF or Spt4/5) is a conserved transcription elongation factor that both inhibits and stimulates transcription elongation in metazoans. InDrosophilaand vertebrates, DSIF together with negative elongation factor (NELF) associates with RNA polymerase II during early elongation and causes RNA polymerase II to pause in the promoter-proximal region of genes. The mechanism of how DSIF establishes pausing is not known. We constructed Spt5 mutant forms of DSIF and tested their capacity to restore promoter-proximal pausing to DSIF-depletedDrosophilanuclear extracts. The C-terminal repeat region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter-proximal pausing. A region encompassing KOW4 and KOW5 of Spt5 is essential for pausing, and mutations in KOW5 specifically shift the location of the pause. RNA cross-linking analysis reveals that KOW5 directly contacts the nascent transcript, and deletion of KOW5 disrupts this interaction. Our results suggest that KOW5 is involved in promoter-proximal pausing through contact with the nascent RNA.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, Pennsylvania 16802 dsg11@psu.edu.
authors.forename: David S
authors.initials: DS
authors.lastname: Gilmour
mesh_terms: D000818:Animals; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000378:metabolism
chemical_list: D002868:Chromosomal Proteins, Non-Histone; C492141:DSIF protein, Drosophila; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C118437:negative elongation factor; C069940:SPT5 transcriptional elongation factor; D012319:RNA Polymerase II
keywords: DSIF; KOW domain; RNA polymerase II; RNA-protein interaction; Spt4/5; mutagenesis; promoter-proximal pausing; transcription elongation factor; transcription regulation