Limits...
Architecture and RNA binding of the human negative elongation factor

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.


Iterative truncation of full-length NELF-AC yields a variant amenable to crystallization and region of the electron density map.(A) Partial digestion of pure full-length NELF-AC with chymotrypsin yields three stable degradation products that were identified as NELF-A N-terminal domain (residues 6–188), NELF-A C-terminal region (residues 248-485) and NELF-C (residues 52–590). The resulting truncated construct NELF-A6-188 C36-590 did not yield diffracting crystals. Shown are SDS-PAGE analyses stained with Coomassie blue. (B) Partial digestion of pure full-length NELF-AC and truncated NELF-A6-188 C36-590 with subtilisin yields the same stable degradation products for NELF-A (residues 6–188) and NELF-C (residues 190–590). (C) The resulting truncated variant NELF-A6-188 C183-590 (‘NELF-AC‘) was successfully used for crystallization. (D) The final 2Fo−Fc electron density map was contoured at 1.5 σ (grey) and the anomalous difference Fourier electron density for the selenomethionine-labeled crystal was contoured at 4.0 σ. The final model for NELF-C helix α4’ is superimposed in stick representation, showing the position of selenium atoms in selenomethionine residues (red) used for phasing. (E) Stereo image of electron density for the crystallized NELF-AC complex. The final 2Fo−Fc electron density map was contoured at 1.5 σ (grey) and the backbone of the complex is shown (cyan NELF-C, red NELF-A).DOI:http://dx.doi.org/10.7554/eLife.14981.003
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940160&req=5

fig1s1: Iterative truncation of full-length NELF-AC yields a variant amenable to crystallization and region of the electron density map.(A) Partial digestion of pure full-length NELF-AC with chymotrypsin yields three stable degradation products that were identified as NELF-A N-terminal domain (residues 6–188), NELF-A C-terminal region (residues 248-485) and NELF-C (residues 52–590). The resulting truncated construct NELF-A6-188 C36-590 did not yield diffracting crystals. Shown are SDS-PAGE analyses stained with Coomassie blue. (B) Partial digestion of pure full-length NELF-AC and truncated NELF-A6-188 C36-590 with subtilisin yields the same stable degradation products for NELF-A (residues 6–188) and NELF-C (residues 190–590). (C) The resulting truncated variant NELF-A6-188 C183-590 (‘NELF-AC‘) was successfully used for crystallization. (D) The final 2Fo−Fc electron density map was contoured at 1.5 σ (grey) and the anomalous difference Fourier electron density for the selenomethionine-labeled crystal was contoured at 4.0 σ. The final model for NELF-C helix α4’ is superimposed in stick representation, showing the position of selenium atoms in selenomethionine residues (red) used for phasing. (E) Stereo image of electron density for the crystallized NELF-AC complex. The final 2Fo−Fc electron density map was contoured at 1.5 σ (grey) and the backbone of the complex is shown (cyan NELF-C, red NELF-A).DOI:http://dx.doi.org/10.7554/eLife.14981.003

Mentions: In a long-standing effort to obtain structural information on the intrinsically flexible NELF complex, we delineated regions in human NELF subunits that form soluble subcomplexes amenable to structural analysis (Figure 1A, Table 1, Figure 1—figure supplement 1, Materials and methods). Bacterial co-expression of NELF subunit variants revealed that the N-terminal region of NELF-A could be co-purified with NELF-C. Limited proteolysis and co-expression analysis with truncated protein variants showed that the N-terminal residues 6–188 of human NELF-A and residues 183–590 of human NELF-C formed a stable subcomplex (‘NELF-AC’) (Figure 1—figure supplement 1B). Purified NELF-AC 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). The structure contained one NELF-AC heterodimer in the asymmetric unit and was refined to a free R-factor of 25.6% at 2.8Å resolution (Table 2). The structure shows very good stereochemistry and lacks only the mobile NELF-A residues 183–188, and NELF-C residues 183–185, 401–402, 445–448, 523, and 564–572.10.7554/eLife.14981.002Figure 1.Primary structure and conservation of human NELF-A and NELF-C.


Architecture and RNA binding of the human negative elongation factor
Iterative truncation of full-length NELF-AC yields a variant amenable to crystallization and region of the electron density map.(A) Partial digestion of pure full-length NELF-AC with chymotrypsin yields three stable degradation products that were identified as NELF-A N-terminal domain (residues 6–188), NELF-A C-terminal region (residues 248-485) and NELF-C (residues 52–590). The resulting truncated construct NELF-A6-188 C36-590 did not yield diffracting crystals. Shown are SDS-PAGE analyses stained with Coomassie blue. (B) Partial digestion of pure full-length NELF-AC and truncated NELF-A6-188 C36-590 with subtilisin yields the same stable degradation products for NELF-A (residues 6–188) and NELF-C (residues 190–590). (C) The resulting truncated variant NELF-A6-188 C183-590 (‘NELF-AC‘) was successfully used for crystallization. (D) The final 2Fo−Fc electron density map was contoured at 1.5 σ (grey) and the anomalous difference Fourier electron density for the selenomethionine-labeled crystal was contoured at 4.0 σ. The final model for NELF-C helix α4’ is superimposed in stick representation, showing the position of selenium atoms in selenomethionine residues (red) used for phasing. (E) Stereo image of electron density for the crystallized NELF-AC complex. The final 2Fo−Fc electron density map was contoured at 1.5 σ (grey) and the backbone of the complex is shown (cyan NELF-C, red NELF-A).DOI:http://dx.doi.org/10.7554/eLife.14981.003
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940160&req=5

fig1s1: Iterative truncation of full-length NELF-AC yields a variant amenable to crystallization and region of the electron density map.(A) Partial digestion of pure full-length NELF-AC with chymotrypsin yields three stable degradation products that were identified as NELF-A N-terminal domain (residues 6–188), NELF-A C-terminal region (residues 248-485) and NELF-C (residues 52–590). The resulting truncated construct NELF-A6-188 C36-590 did not yield diffracting crystals. Shown are SDS-PAGE analyses stained with Coomassie blue. (B) Partial digestion of pure full-length NELF-AC and truncated NELF-A6-188 C36-590 with subtilisin yields the same stable degradation products for NELF-A (residues 6–188) and NELF-C (residues 190–590). (C) The resulting truncated variant NELF-A6-188 C183-590 (‘NELF-AC‘) was successfully used for crystallization. (D) The final 2Fo−Fc electron density map was contoured at 1.5 σ (grey) and the anomalous difference Fourier electron density for the selenomethionine-labeled crystal was contoured at 4.0 σ. The final model for NELF-C helix α4’ is superimposed in stick representation, showing the position of selenium atoms in selenomethionine residues (red) used for phasing. (E) Stereo image of electron density for the crystallized NELF-AC complex. The final 2Fo−Fc electron density map was contoured at 1.5 σ (grey) and the backbone of the complex is shown (cyan NELF-C, red NELF-A).DOI:http://dx.doi.org/10.7554/eLife.14981.003
Mentions: In a long-standing effort to obtain structural information on the intrinsically flexible NELF complex, we delineated regions in human NELF subunits that form soluble subcomplexes amenable to structural analysis (Figure 1A, Table 1, Figure 1—figure supplement 1, Materials and methods). Bacterial co-expression of NELF subunit variants revealed that the N-terminal region of NELF-A could be co-purified with NELF-C. Limited proteolysis and co-expression analysis with truncated protein variants showed that the N-terminal residues 6–188 of human NELF-A and residues 183–590 of human NELF-C formed a stable subcomplex (‘NELF-AC’) (Figure 1—figure supplement 1B). Purified NELF-AC 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). The structure contained one NELF-AC heterodimer in the asymmetric unit and was refined to a free R-factor of 25.6% at 2.8Å resolution (Table 2). The structure shows very good stereochemistry and lacks only the mobile NELF-A residues 183–188, and NELF-C residues 183–185, 401–402, 445–448, 523, and 564–572.10.7554/eLife.14981.002Figure 1.Primary structure and conservation of human NELF-A and NELF-C.

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.