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Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex.

The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, A to E. NELF-A has sequence similarity to hepatitis delta antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding activity is critical for NELF function. To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we identified the B, C, and D proteins of human NELF. NELF-B is identical to COBRA1, recently reported to associate with the product of breast cancer susceptibility gene BRCA1. NELF-C and NELF-D are highly related or identical to the protein called TH1, of unknown function. NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression of these four proteins in insect cells resulted in the reconstitution of functionally active NELF. Detailed analyses using mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding and for transcriptional pausing. This study defines several important protein-protein interactions and opens the way for understanding the mechanism of DSIF- and NELF-induced transcriptional pausing.

Pubmed ID: 12612062

Authors

  • Narita T
  • Yamaguchi Y
  • Yano K
  • Sugimoto S
  • Chanarat S
  • Wada T
  • Kim DK
  • Hasegawa J
  • Omori M
  • Inukai N
  • Endoh M
  • Yamada T
  • Handa H

Journal

Molecular and cellular biology

Publication Data

March 3, 2003

Associated Grants

None

Mesh Terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • Evolution, Molecular
  • Humans
  • Macromolecular Substances
  • Models, Biological
  • Molecular Sequence Data
  • Nuclear Proteins
  • Peptide Chain Elongation, Translational
  • Positive Transcriptional Elongation Factor B
  • Protein Interaction Mapping
  • Protein Subunits
  • Protein-Serine-Threonine Kinases
  • RNA Polymerase II
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Sequence Analysis, Protein
  • Structure-Activity Relationship
  • Transcription Factors
  • Transcriptional Elongation Factors