Architecture and RNA binding of the human negative elongation factor.
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.
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Seychelle M
SM
Vos
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.|Gene Center Munich, Ludwig-Maximilians-Universität München, Munich, Germany.|Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.
David
D
Pöllmann
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Livia
L
Caizzi
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Katharina B
KB
Hofmann
Gene Center Munich, Ludwig-Maximilians-Universität München, Munich, Germany.|Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.
Pascaline
P
Rombaut
Gene Center Munich, Ludwig-Maximilians-Universität München, Munich, Germany.|Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.
Tomasz
T
Zimniak
Gene Center Munich, Ludwig-Maximilians-Universität München, Munich, Germany.|Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.
Franz
F
Herzog
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Patrick
0000-0001-5454-7755
P
Cramer
D018360:Crystallography, X-Ray; D005786:Gene Expression Regulation; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic
C411985:NSMF protein, human; D014157:Transcription Factors; D012313:RNA
RNA polymerase II; biochemistry; biophysics; gene regulation; gene transcription; human; promoter-proximal pausing; structural biology