Molecular characterization of Drosophila NELF.
NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Here, further characterization of Drosophila NELF is provided. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N- and C-terminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and beta1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila.
Wu
Chwen-Huey
CH
Department of Biochemistry and Molecular Biology, Center for Gene Regulation, The Pennsylvania State University University Park, PA 16802, USA.
Lee
Chanhyo
C
Fan
Ruopeng
R
Smith
Meghan J
MJ
Yamaguchi
Yuki
Y
Handa
Hiroshi
H
Gilmour
David S
DS
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics; D002843:Chromatin / Q000737:chemistry; D002875:Chromosomes / Q000737:chemistry; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004330:Drosophila / Q000166:cytology / Q000235:genetics*; D029721:Drosophila Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D014404:Tubulin / Q000235:genetics
D002843:Chromatin; C492141:DSIF protein, Drosophila; D029721:Drosophila Proteins; D018840:HSP70 Heat-Shock Proteins; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D021122:Protein Subunits; D014157:Transcription Factors; D014404:Tubulin; C118437:negative elongation factor; D004004:Dichlororibofuranosylbenzimidazole
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