Abstract
Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.
Highlights
Transcription of eukaryotic protein-coding genes by RNA polymerase II (Pol II) is regulated during the initiation phase (Hahn and Young, 2011; Sainsbury et al, 2015) and during elongation (Jonkers and Lis, 2015; Li and Gilmour, 2011; Yamaguchi et al, 2013)
Purified NELFAC could be crystallized by vapor diffusion, and the X-ray structure was solved by single isomorphous replacement with anomalous scattering (SIRAS) (Figure 1—figure supplement 1C–E, Materials and methods)
Deciphering the mechanism of promoter-proximal Pol II pausing is essential for understanding gene regulation and requires structural information of Pol II elongation complexes bound by DRB sensitivity-inducing factor (DSIF), negative elongation factor (NELF), and positive transcription elongation factor b (P-TEFb)
Summary
Transcription of eukaryotic protein-coding genes by RNA polymerase II (Pol II) is regulated during the initiation phase (Hahn and Young, 2011; Sainsbury et al, 2015) and during elongation (Jonkers and Lis, 2015; Li and Gilmour, 2011; Yamaguchi et al, 2013). For many metazoan genes, elongating Pol II pauses near the promoter, about 20-60 base pairs downstream of the transcription start site (TSS) (Kwak and Lis, 2013). Such promoter-proximal pausing is a key event during post-initiation regulation of transcription (Muse et al, 2007; Zeitlinger et al, 2007). Promoter-proximal pausing employs the negative elongation factor (NELF) (Pagano et al, 2014; Yamaguchi et al, 1999a), which comprises the four subunits NELF-A, -B, -C (or its variant -D, which lacks nine N-terminal amino acid residues), and -E (Narita et al, 2003)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
