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Molecular and Cellular Biology, March 2003, p. 1863-1873, Vol. 23, No. 6
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.6.1863-1873.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Human Transcription Elongation Factor NELF: Identification of Novel Subunits and Reconstitution of the Functionally Active Complex

Takashi Narita,1 Yuki Yamaguchi,1,2 Keiichi Yano,3 Seiji Sugimoto,3 Sittinan Chanarat,1 Tadashi Wada,1 Dong-ki Kim,1 Jun Hasegawa,1 Masashi Omori,1 Naoto Inukai,1 Masaki Endoh,1 Tomoko Yamada,1 and Hiroshi Handa4*

Graduate School of Bioscience and Biotechnology,1 Frontier Collaborative Research Center, Tokyo Institute of Technology,4 PRESTO, Japan Science and Technology Corporation, Yokohama 226-8503,2 Tokyo Research Laboratories, Kyowa Hakko Kogyo Company, Tokyo 194-8533, Japan3

Received 8 October 2002/ Returned for modification 25 November 2002/ Accepted 23 December 2002

The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-ß-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.

* Corresponding author. Mailing address: Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8503, Japan. Phone: 81-45-924-5872. Fax: 81-45-924-5145. E-mail: hhanda{at}bio.titech.ac.jp.

Molecular and Cellular Biology, March 2003, p. 1863-1873, Vol. 23, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.6.1863-1873.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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