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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.


Purity of NELF truncation constructs and DNA binding.(A) SDS-PAGE of purified NELF truncations, NELF-B, and NELF-BE (1–138). Protein (0.9 µg) was run on a 4–12% gel and stained with Coomassie blue. Molecular weight in kDa is indicated on the left side of the gel. (B) Binding of the WT NELF tetramer, NELF ∆RRM, or NELF-ABC to the 60% GC content ssDNA as determined by fluorescence anisotropy. Error bars reflect the standard deviation from three experimental replicates. (C) SDS-PAGE of purified NELF constructs containing patch mutated NELF-C. Protein (0.9 µg) was run on a gradient 4–12% SDS-PAGE and stained with Coomassie blue. Molecular weight is indicated on the left side of the gel. (D–F) Binding of the WT NELF tetramer and a NELF tetramer with patch mutated NELF-C to the 60% GC content ssDNA as determined by fluorescence anisotropy. Error bars reflect the standard deviation from three experimental replicates. Darker shades of blue indicate the WT protein constructs whereas lighter shades of blue indicate the NELF-C patch mutated variant. Error bars reflect the standard deviation from three experimental replicates. (D) NELF tetramer (E) NELF ∆RRM (F) NELF-ABC.DOI:http://dx.doi.org/10.7554/eLife.14981.018
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fig6s1: Purity of NELF truncation constructs and DNA binding.(A) SDS-PAGE of purified NELF truncations, NELF-B, and NELF-BE (1–138). Protein (0.9 µg) was run on a 4–12% gel and stained with Coomassie blue. Molecular weight in kDa is indicated on the left side of the gel. (B) Binding of the WT NELF tetramer, NELF ∆RRM, or NELF-ABC to the 60% GC content ssDNA as determined by fluorescence anisotropy. Error bars reflect the standard deviation from three experimental replicates. (C) SDS-PAGE of purified NELF constructs containing patch mutated NELF-C. Protein (0.9 µg) was run on a gradient 4–12% SDS-PAGE and stained with Coomassie blue. Molecular weight is indicated on the left side of the gel. (D–F) Binding of the WT NELF tetramer and a NELF tetramer with patch mutated NELF-C to the 60% GC content ssDNA as determined by fluorescence anisotropy. Error bars reflect the standard deviation from three experimental replicates. Darker shades of blue indicate the WT protein constructs whereas lighter shades of blue indicate the NELF-C patch mutated variant. Error bars reflect the standard deviation from three experimental replicates. (D) NELF tetramer (E) NELF ∆RRM (F) NELF-ABC.DOI:http://dx.doi.org/10.7554/eLife.14981.018

Mentions: We next addressed whether NELF-AC associates with nucleic acids while residing in the NELF tetramer. The NELF-E RRM is reported to bind RNA in the mid nanomolar to micromolar range (Pagano et al., 2014; Rao et al., 2006) and thus could mask nucleic acid interactions by other subunits in our binding assays. To aid data interpretation, we generated NELF variants that lack the NELF-E RRM or NELF-E entirely. We used our crosslinking and limited proteolysis experiments to generate a NELF-E N-terminal fragment that stably binds NELF-B, but lacks the RRM (NELF-E residues 1–138). The WT NELF tetramer, NELF ∆RRM, and NELF-ABC were overexpressed in insect cells and purified to homogeneity (Materials and methods, Figure 6A, Figure 6—figure supplement 1A).10.7554/eLife.14981.017Figure 6.NELF-AC associates with RNA in context of the NELF tetramer.


Architecture and RNA binding of the human negative elongation factor
Purity of NELF truncation constructs and DNA binding.(A) SDS-PAGE of purified NELF truncations, NELF-B, and NELF-BE (1–138). Protein (0.9 µg) was run on a 4–12% gel and stained with Coomassie blue. Molecular weight in kDa is indicated on the left side of the gel. (B) Binding of the WT NELF tetramer, NELF ∆RRM, or NELF-ABC to the 60% GC content ssDNA as determined by fluorescence anisotropy. Error bars reflect the standard deviation from three experimental replicates. (C) SDS-PAGE of purified NELF constructs containing patch mutated NELF-C. Protein (0.9 µg) was run on a gradient 4–12% SDS-PAGE and stained with Coomassie blue. Molecular weight is indicated on the left side of the gel. (D–F) Binding of the WT NELF tetramer and a NELF tetramer with patch mutated NELF-C to the 60% GC content ssDNA as determined by fluorescence anisotropy. Error bars reflect the standard deviation from three experimental replicates. Darker shades of blue indicate the WT protein constructs whereas lighter shades of blue indicate the NELF-C patch mutated variant. Error bars reflect the standard deviation from three experimental replicates. (D) NELF tetramer (E) NELF ∆RRM (F) NELF-ABC.DOI:http://dx.doi.org/10.7554/eLife.14981.018
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fig6s1: Purity of NELF truncation constructs and DNA binding.(A) SDS-PAGE of purified NELF truncations, NELF-B, and NELF-BE (1–138). Protein (0.9 µg) was run on a 4–12% gel and stained with Coomassie blue. Molecular weight in kDa is indicated on the left side of the gel. (B) Binding of the WT NELF tetramer, NELF ∆RRM, or NELF-ABC to the 60% GC content ssDNA as determined by fluorescence anisotropy. Error bars reflect the standard deviation from three experimental replicates. (C) SDS-PAGE of purified NELF constructs containing patch mutated NELF-C. Protein (0.9 µg) was run on a gradient 4–12% SDS-PAGE and stained with Coomassie blue. Molecular weight is indicated on the left side of the gel. (D–F) Binding of the WT NELF tetramer and a NELF tetramer with patch mutated NELF-C to the 60% GC content ssDNA as determined by fluorescence anisotropy. Error bars reflect the standard deviation from three experimental replicates. Darker shades of blue indicate the WT protein constructs whereas lighter shades of blue indicate the NELF-C patch mutated variant. Error bars reflect the standard deviation from three experimental replicates. (D) NELF tetramer (E) NELF ∆RRM (F) NELF-ABC.DOI:http://dx.doi.org/10.7554/eLife.14981.018
Mentions: We next addressed whether NELF-AC associates with nucleic acids while residing in the NELF tetramer. The NELF-E RRM is reported to bind RNA in the mid nanomolar to micromolar range (Pagano et al., 2014; Rao et al., 2006) and thus could mask nucleic acid interactions by other subunits in our binding assays. To aid data interpretation, we generated NELF variants that lack the NELF-E RRM or NELF-E entirely. We used our crosslinking and limited proteolysis experiments to generate a NELF-E N-terminal fragment that stably binds NELF-B, but lacks the RRM (NELF-E residues 1–138). The WT NELF tetramer, NELF ∆RRM, and NELF-ABC were overexpressed in insect cells and purified to homogeneity (Materials and methods, Figure 6A, Figure 6—figure supplement 1A).10.7554/eLife.14981.017Figure 6.NELF-AC associates with RNA in context of the NELF tetramer.

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.