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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.


NELF-B association with ssRNA, ssDNA and TAR RNA stem loop.(A) Binding of NELF-B (light red) or NELF-BE (1–138) (red) to the 60% GC RNA as determined by fluorescence anisotropy. NELF-AC (dark red) (Figure 5A) binding to the same RNA is shown as a reference. Error bars reflect the standard deviation from three experimental replicates. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3. (B) Binding of NELF-B (cyan) or NELF-BE (1–138) (sky blue) to the 60% GC DNA as determined by fluorescence anisotropy. NELF-AC (dark blue) (Figure 5A) binding is shown as a reference. Error bars reflect the standard deviation from three experimental replicates. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3. (C) 2D structure of TAR RNA stem loop region used for fluorescence anisotropy experiments presented in (D). Dots indicate hydrogen bonds between bases. Lines represent the phosphate backbone. RNA was labeled with a 5’ FAM label. (D) Binding of the NELF tetramer (dark purple), NELF ∆RRM (orchid), NELF-ABC (thistle), NELF-BE (1–138) (medium purple), and NELF-AC (light purple) to the TAR RNA stem loop. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3.DOI:http://dx.doi.org/10.7554/eLife.14981.019
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fig7: NELF-B association with ssRNA, ssDNA and TAR RNA stem loop.(A) Binding of NELF-B (light red) or NELF-BE (1–138) (red) to the 60% GC RNA as determined by fluorescence anisotropy. NELF-AC (dark red) (Figure 5A) binding to the same RNA is shown as a reference. Error bars reflect the standard deviation from three experimental replicates. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3. (B) Binding of NELF-B (cyan) or NELF-BE (1–138) (sky blue) to the 60% GC DNA as determined by fluorescence anisotropy. NELF-AC (dark blue) (Figure 5A) binding is shown as a reference. Error bars reflect the standard deviation from three experimental replicates. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3. (C) 2D structure of TAR RNA stem loop region used for fluorescence anisotropy experiments presented in (D). Dots indicate hydrogen bonds between bases. Lines represent the phosphate backbone. RNA was labeled with a 5’ FAM label. (D) Binding of the NELF tetramer (dark purple), NELF ∆RRM (orchid), NELF-ABC (thistle), NELF-BE (1–138) (medium purple), and NELF-AC (light purple) to the TAR RNA stem loop. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3.DOI:http://dx.doi.org/10.7554/eLife.14981.019

Mentions: Curve fitting data for NELF RNA and DNA binding as determined by fluorescence anisotropy. Fluorescence anisotropy data found in Figures 5–7 were fit with a single site binding model where possible (Materials and methods). Apparent disassociation constants (Kd,app), R2, and Bmax (maximum anisotropy) values with error where applicable for the 60% ssRNA and DNA substrate as well as the TAR RNA are shown. NA means fitting was not applicable.


Architecture and RNA binding of the human negative elongation factor
NELF-B association with ssRNA, ssDNA and TAR RNA stem loop.(A) Binding of NELF-B (light red) or NELF-BE (1–138) (red) to the 60% GC RNA as determined by fluorescence anisotropy. NELF-AC (dark red) (Figure 5A) binding to the same RNA is shown as a reference. Error bars reflect the standard deviation from three experimental replicates. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3. (B) Binding of NELF-B (cyan) or NELF-BE (1–138) (sky blue) to the 60% GC DNA as determined by fluorescence anisotropy. NELF-AC (dark blue) (Figure 5A) binding is shown as a reference. Error bars reflect the standard deviation from three experimental replicates. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3. (C) 2D structure of TAR RNA stem loop region used for fluorescence anisotropy experiments presented in (D). Dots indicate hydrogen bonds between bases. Lines represent the phosphate backbone. RNA was labeled with a 5’ FAM label. (D) Binding of the NELF tetramer (dark purple), NELF ∆RRM (orchid), NELF-ABC (thistle), NELF-BE (1–138) (medium purple), and NELF-AC (light purple) to the TAR RNA stem loop. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3.DOI:http://dx.doi.org/10.7554/eLife.14981.019
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fig7: NELF-B association with ssRNA, ssDNA and TAR RNA stem loop.(A) Binding of NELF-B (light red) or NELF-BE (1–138) (red) to the 60% GC RNA as determined by fluorescence anisotropy. NELF-AC (dark red) (Figure 5A) binding to the same RNA is shown as a reference. Error bars reflect the standard deviation from three experimental replicates. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3. (B) Binding of NELF-B (cyan) or NELF-BE (1–138) (sky blue) to the 60% GC DNA as determined by fluorescence anisotropy. NELF-AC (dark blue) (Figure 5A) binding is shown as a reference. Error bars reflect the standard deviation from three experimental replicates. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3. (C) 2D structure of TAR RNA stem loop region used for fluorescence anisotropy experiments presented in (D). Dots indicate hydrogen bonds between bases. Lines represent the phosphate backbone. RNA was labeled with a 5’ FAM label. (D) Binding of the NELF tetramer (dark purple), NELF ∆RRM (orchid), NELF-ABC (thistle), NELF-BE (1–138) (medium purple), and NELF-AC (light purple) to the TAR RNA stem loop. Data were fit with a single site binding model. Apparent Kd values are reported in Table 3.DOI:http://dx.doi.org/10.7554/eLife.14981.019
Mentions: Curve fitting data for NELF RNA and DNA binding as determined by fluorescence anisotropy. Fluorescence anisotropy data found in Figures 5–7 were fit with a single site binding model where possible (Materials and methods). Apparent disassociation constants (Kd,app), R2, and Bmax (maximum anisotropy) values with error where applicable for the 60% ssRNA and DNA substrate as well as the TAR RNA are shown. NA means fitting was not applicable.

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.