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Architecture and RNA binding of the human negative elongation factor

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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.

Doi:: http://dx.doi.org/10.7554/eLife.14981.001

No MeSH data available.


Related in: MedlinePlus

Crystal structure of human NELF-AC complex.(A) Ribbon model of NELF-AC with NELF-A in red and NELF-C in cyan. N- and C-termini, mobile regions, and truncated regions are indicated by dashed lines. The two views are related by a 180° rotation around the vertical axis. Curved lines marked 'H1–H3' demarcate alpha helices involved in heat repeats 1–3 (1: 14’, 15’; 2: 16’, 17’; 3: 18’, 19’). Alpha helices are named as in Figure 1B. All crystallography figures were rendered with Pymol (PyMOL, 2002). (B) NELF-A N-terminal domain enlarged and rotated 60° around the horizontal axis relative to 'bottom view' (Figure 2A). (C) Detailed view of invariant NELF-A residues W24 and W89 and surrounding residues (stick model) interacting with the NELF-C surface. NELF-C surface conservation colored according to Figure 1B. The view is rotated by 90° around the vertical axis relative to 'bottom view' (Figure 2A). (D) Detailed view of NELF-A helices α7 and α8 (stick model, residues 138–154) surrounded by NELF-C. NELF-C surface conservation is colored according to Figure 1B. The view is rotated 60° around the horizontal axis relative to 'bottom view' (Figure 2A).DOI:http://dx.doi.org/10.7554/eLife.14981.008
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fig2: Crystal structure of human NELF-AC complex.(A) Ribbon model of NELF-AC with NELF-A in red and NELF-C in cyan. N- and C-termini, mobile regions, and truncated regions are indicated by dashed lines. The two views are related by a 180° rotation around the vertical axis. Curved lines marked 'H1–H3' demarcate alpha helices involved in heat repeats 1–3 (1: 14’, 15’; 2: 16’, 17’; 3: 18’, 19’). Alpha helices are named as in Figure 1B. All crystallography figures were rendered with Pymol (PyMOL, 2002). (B) NELF-A N-terminal domain enlarged and rotated 60° around the horizontal axis relative to 'bottom view' (Figure 2A). (C) Detailed view of invariant NELF-A residues W24 and W89 and surrounding residues (stick model) interacting with the NELF-C surface. NELF-C surface conservation colored according to Figure 1B. The view is rotated by 90° around the vertical axis relative to 'bottom view' (Figure 2A). (D) Detailed view of NELF-A helices α7 and α8 (stick model, residues 138–154) surrounded by NELF-C. NELF-C surface conservation is colored according to Figure 1B. The view is rotated 60° around the horizontal axis relative to 'bottom view' (Figure 2A).DOI:http://dx.doi.org/10.7554/eLife.14981.008

Mentions: The structure of human NELF-AC reveals a novel fold and an extended interface between the two NELF subunits (Figures 1B, 2). NELF-C adopts a horseshoe-like structure (Figure 2A). NELF-C consists of 22 α-helices (α1’-α22’) and a small two-stranded β-sheet (β1’-β2’, residues 367–379) that protrudes from the surface. The C-terminal half of NELF-C (helices α14’-α19’) forms three HEAT repeats (H1-H3). The HEAT repeat region shows structural similarity (Holm and Rosenstrom, 2010) to the C-terminal repeat domain (CTD)-interacting domain (CID) (Meinhart and Cramer, 2004) and the polyadenylation factor symplekin (Xiang et al., 2010). Despite the presence of a CID-like fold, NELF-AC did not show significant binding to CTD diheptad peptides carrying phosphorylations at CTD residues serine-2, serine-5, serine-2 and serine-5, or a consensus non-phosphorylated CTD diheptad peptide (not shown). Subunit NELF-A forms a highly conserved helical ‘N-terminal domain’ (helices α1–α5, residues 6–110) that resembles (Holm and Rosenstrom, 2010) the fold of the HIV integrase-binding domain present in human PC4 and SFRS1-interacting protein (PSIP1) (Cherepanov et al., 2005) (Figure 2B). This domain is followed by an ‘extended region’ in NELF-A that forms four additional helices (helices α6-α9, residues 111–182) arrayed around the NELF-C horseshoe (Figures 1B, 2A).10.7554/eLife.14981.008Figure 2.Crystal structure of human NELF-AC complex.


Architecture and RNA binding of the human negative elongation factor
Crystal structure of human NELF-AC complex.(A) Ribbon model of NELF-AC with NELF-A in red and NELF-C in cyan. N- and C-termini, mobile regions, and truncated regions are indicated by dashed lines. The two views are related by a 180° rotation around the vertical axis. Curved lines marked 'H1–H3' demarcate alpha helices involved in heat repeats 1–3 (1: 14’, 15’; 2: 16’, 17’; 3: 18’, 19’). Alpha helices are named as in Figure 1B. All crystallography figures were rendered with Pymol (PyMOL, 2002). (B) NELF-A N-terminal domain enlarged and rotated 60° around the horizontal axis relative to 'bottom view' (Figure 2A). (C) Detailed view of invariant NELF-A residues W24 and W89 and surrounding residues (stick model) interacting with the NELF-C surface. NELF-C surface conservation colored according to Figure 1B. The view is rotated by 90° around the vertical axis relative to 'bottom view' (Figure 2A). (D) Detailed view of NELF-A helices α7 and α8 (stick model, residues 138–154) surrounded by NELF-C. NELF-C surface conservation is colored according to Figure 1B. The view is rotated 60° around the horizontal axis relative to 'bottom view' (Figure 2A).DOI:http://dx.doi.org/10.7554/eLife.14981.008
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fig2: Crystal structure of human NELF-AC complex.(A) Ribbon model of NELF-AC with NELF-A in red and NELF-C in cyan. N- and C-termini, mobile regions, and truncated regions are indicated by dashed lines. The two views are related by a 180° rotation around the vertical axis. Curved lines marked 'H1–H3' demarcate alpha helices involved in heat repeats 1–3 (1: 14’, 15’; 2: 16’, 17’; 3: 18’, 19’). Alpha helices are named as in Figure 1B. All crystallography figures were rendered with Pymol (PyMOL, 2002). (B) NELF-A N-terminal domain enlarged and rotated 60° around the horizontal axis relative to 'bottom view' (Figure 2A). (C) Detailed view of invariant NELF-A residues W24 and W89 and surrounding residues (stick model) interacting with the NELF-C surface. NELF-C surface conservation colored according to Figure 1B. The view is rotated by 90° around the vertical axis relative to 'bottom view' (Figure 2A). (D) Detailed view of NELF-A helices α7 and α8 (stick model, residues 138–154) surrounded by NELF-C. NELF-C surface conservation is colored according to Figure 1B. The view is rotated 60° around the horizontal axis relative to 'bottom view' (Figure 2A).DOI:http://dx.doi.org/10.7554/eLife.14981.008
Mentions: The structure of human NELF-AC reveals a novel fold and an extended interface between the two NELF subunits (Figures 1B, 2). NELF-C adopts a horseshoe-like structure (Figure 2A). NELF-C consists of 22 α-helices (α1’-α22’) and a small two-stranded β-sheet (β1’-β2’, residues 367–379) that protrudes from the surface. The C-terminal half of NELF-C (helices α14’-α19’) forms three HEAT repeats (H1-H3). The HEAT repeat region shows structural similarity (Holm and Rosenstrom, 2010) to the C-terminal repeat domain (CTD)-interacting domain (CID) (Meinhart and Cramer, 2004) and the polyadenylation factor symplekin (Xiang et al., 2010). Despite the presence of a CID-like fold, NELF-AC did not show significant binding to CTD diheptad peptides carrying phosphorylations at CTD residues serine-2, serine-5, serine-2 and serine-5, or a consensus non-phosphorylated CTD diheptad peptide (not shown). Subunit NELF-A forms a highly conserved helical ‘N-terminal domain’ (helices α1–α5, residues 6–110) that resembles (Holm and Rosenstrom, 2010) the fold of the HIV integrase-binding domain present in human PC4 and SFRS1-interacting protein (PSIP1) (Cherepanov et al., 2005) (Figure 2B). This domain is followed by an ‘extended region’ in NELF-A that forms four additional helices (helices α6-α9, residues 111–182) arrayed around the NELF-C horseshoe (Figures 1B, 2A).10.7554/eLife.14981.008Figure 2.Crystal structure of human NELF-AC complex.

View Article: PubMed Central - PubMed

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.

Doi:: http://dx.doi.org/10.7554/eLife.14981.001

No MeSH data available.


Related in: MedlinePlus