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

Abstract

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.

• Authors: Takashi Narita, Yuki Yamaguchi, Keiichi Yano, Seiji Sugimoto, Sittinan Chanarat, Tadashi Wada, Dong-ki Kim, Jun Hasegawa, Masashi Omori, Naoto Inukai, Masaki Endoh, Tomoko Yamada, Hiroshi Handa
• Journal: Molecular and cellular biology (Mol Cell Biol) Vol. 23 Issue 6 Pg. 1863-73 (Mar 2003) ISSN: 0270-7306 [Print] United States
• PMID: 12612062 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Macromolecular Substances
• Nuclear Proteins
• Protein Subunits
• Repressor Proteins
• SUPT4H1 protein, human
• SUPT5H protein, human
• Saccharomyces cerevisiae Proteins
• Transcription Factors
• Transcriptional Elongation Factors
• negative elongation factor
• Positive Transcriptional Elongation Factor B
• Protein Serine-Threonine Kinases
• RNA Polymerase II

Topics

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