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.
Narita
Takashi
T
Graduate School of Bioscience and Biotechnolog, Tokyo Institute of Technology, 4259 Nagatsuka, Yokohama 226-8503, Japan.
Yamaguchi
Yuki
Y
Yano
Keiichi
K
Sugimoto
Seiji
S
Chanarat
Sittinan
S
Wada
Tadashi
T
Kim
Dong-ki
DK
Hasegawa
Jun
J
Omori
Masashi
M
Inukai
Naoto
N
Endoh
Masaki
M
Yamada
Tomoko
T
Handa
Hiroshi
H
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D006801:Humans; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational / Q000502:physiology*; D042925:Positive Transcriptional Elongation Factor B; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D046911:Macromolecular Substances; D009687:Nuclear Proteins; D021122:Protein Subunits; D012097:Repressor Proteins; C481011:SUPT4H1 protein, human; C112224:SUPT5H protein, human; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C118437:negative elongation factor; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases; D012319:RNA Polymerase II