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.
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
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
Mol. Cell. Biol.
Date: Mar. 01, 2003
PubMed ID: 12612062
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