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Molecular and Cellular Biology, May 2002, p. 2918-2927, Vol. 22, No. 9
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.9.2918-2927.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Evidence that Negative Elongation Factor Represses Transcription Elongation through Binding to a DRB Sensitivity-Inducing Factor/RNA Polymerase II Complex and RNA

Yuki Yamaguchi,1,2 Naoto Inukai,1 Takashi Narita,1 Tadashi Wada,1 and Hiroshi Handa3*

Faculty of Bioscience and Biotechnology,1 Frontier Collaborative Research Center, Tokyo Institute of Technology,3 PRESTO, Japan Science and Technology Corporation, Yokohama, Japan2

Received 7 November 2001/ Returned for modification 7 January 2002/ Accepted 28 January 2002

Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.

* 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, May 2002, p. 2918-2927, Vol. 22, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.9.2918-2927.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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