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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.


Related in: MedlinePlus

Fluorescence anisotropy controls.NELF-AC DNA (A) and RNA (B) binding detected by fluorescence anisotropy in the presence or absence of tRNA. Baker’s yeast tRNA was added to a final concentration of 5 µg/mL (approx. 200 nM, 100x greater concentration than substrate RNA) to determine whether NELF-AC nucleic acid binding activity is specific. The DNA and RNA substrates are the same used in Figure 4A,B. Error bars are representative of three experimental replicates. (C) Binding of WT NELF-AC to 10 nM of fluorescently labeled ssRNA and ssDNA derived from natural sequences of promoter-proximal regions of paused genes junB and c-fos (Materials and methods) as monitored by the change in relative fluorescence anisotropy. Inset: Schematic of the presence of single-stranded nucleic acids (ssRNA, ssDNA) in the promoter-proximally paused Pol II elongation complex. Error bars are representative of three experimental replicates.DOI:http://dx.doi.org/10.7554/eLife.14981.015
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fig5s1: Fluorescence anisotropy controls.NELF-AC DNA (A) and RNA (B) binding detected by fluorescence anisotropy in the presence or absence of tRNA. Baker’s yeast tRNA was added to a final concentration of 5 µg/mL (approx. 200 nM, 100x greater concentration than substrate RNA) to determine whether NELF-AC nucleic acid binding activity is specific. The DNA and RNA substrates are the same used in Figure 4A,B. Error bars are representative of three experimental replicates. (C) Binding of WT NELF-AC to 10 nM of fluorescently labeled ssRNA and ssDNA derived from natural sequences of promoter-proximal regions of paused genes junB and c-fos (Materials and methods) as monitored by the change in relative fluorescence anisotropy. Inset: Schematic of the presence of single-stranded nucleic acids (ssRNA, ssDNA) in the promoter-proximally paused Pol II elongation complex. Error bars are representative of three experimental replicates.DOI:http://dx.doi.org/10.7554/eLife.14981.015

Mentions: The positively charged patches of NELF-AC suggested that the subcomplex may associate with nucleic acid. To investigate this, we used fluorescence anisotropy titration assays (Figure 5, Materials and methods). We first assessed NELF-AC binding to 25-nt, single-stranded (ss) DNA and ssRNA oligonucleotides bearing a 5’ FAM label. Two random sequences with either 44% or 60% GC content were employed. Interestingly, we detected moderate binding of NELF-AC to the ssDNA and ssRNA with 60% GC content. Fitting the resulting binding curves by linear regression analysis gave apparent Kd’s in the low micromolar range (Figure 5A, Table 3). The addition of competitor tRNA did not affect NELF-AC association with the 60% GC ssDNA/ssRNA indicating that the interaction is specific (Figure 5—figure supplement 1A). In contrast, we found that the 44% GC content RNA failed to associate significantly with the NELF-AC complex (Figure 5B). Additionally, NELF-AC did not associate with nucleic acid duplexes composed of the 60% GC sequence (DNA or DNA-RNA hybrids, not shown), suggesting that RNA and DNA binding by the subcomplex may be sequence and structure dependent.10.7554/eLife.14981.014Figure 5.NELF-AC binds single-stranded nucleic acids.


Architecture and RNA binding of the human negative elongation factor
Fluorescence anisotropy controls.NELF-AC DNA (A) and RNA (B) binding detected by fluorescence anisotropy in the presence or absence of tRNA. Baker’s yeast tRNA was added to a final concentration of 5 µg/mL (approx. 200 nM, 100x greater concentration than substrate RNA) to determine whether NELF-AC nucleic acid binding activity is specific. The DNA and RNA substrates are the same used in Figure 4A,B. Error bars are representative of three experimental replicates. (C) Binding of WT NELF-AC to 10 nM of fluorescently labeled ssRNA and ssDNA derived from natural sequences of promoter-proximal regions of paused genes junB and c-fos (Materials and methods) as monitored by the change in relative fluorescence anisotropy. Inset: Schematic of the presence of single-stranded nucleic acids (ssRNA, ssDNA) in the promoter-proximally paused Pol II elongation complex. Error bars are representative of three experimental replicates.DOI:http://dx.doi.org/10.7554/eLife.14981.015
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Related In: Results  -  Collection

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fig5s1: Fluorescence anisotropy controls.NELF-AC DNA (A) and RNA (B) binding detected by fluorescence anisotropy in the presence or absence of tRNA. Baker’s yeast tRNA was added to a final concentration of 5 µg/mL (approx. 200 nM, 100x greater concentration than substrate RNA) to determine whether NELF-AC nucleic acid binding activity is specific. The DNA and RNA substrates are the same used in Figure 4A,B. Error bars are representative of three experimental replicates. (C) Binding of WT NELF-AC to 10 nM of fluorescently labeled ssRNA and ssDNA derived from natural sequences of promoter-proximal regions of paused genes junB and c-fos (Materials and methods) as monitored by the change in relative fluorescence anisotropy. Inset: Schematic of the presence of single-stranded nucleic acids (ssRNA, ssDNA) in the promoter-proximally paused Pol II elongation complex. Error bars are representative of three experimental replicates.DOI:http://dx.doi.org/10.7554/eLife.14981.015
Mentions: The positively charged patches of NELF-AC suggested that the subcomplex may associate with nucleic acid. To investigate this, we used fluorescence anisotropy titration assays (Figure 5, Materials and methods). We first assessed NELF-AC binding to 25-nt, single-stranded (ss) DNA and ssRNA oligonucleotides bearing a 5’ FAM label. Two random sequences with either 44% or 60% GC content were employed. Interestingly, we detected moderate binding of NELF-AC to the ssDNA and ssRNA with 60% GC content. Fitting the resulting binding curves by linear regression analysis gave apparent Kd’s in the low micromolar range (Figure 5A, Table 3). The addition of competitor tRNA did not affect NELF-AC association with the 60% GC ssDNA/ssRNA indicating that the interaction is specific (Figure 5—figure supplement 1A). In contrast, we found that the 44% GC content RNA failed to associate significantly with the NELF-AC complex (Figure 5B). Additionally, NELF-AC did not associate with nucleic acid duplexes composed of the 60% GC sequence (DNA or DNA-RNA hybrids, not shown), suggesting that RNA and DNA binding by the subcomplex may be sequence and structure dependent.10.7554/eLife.14981.014Figure 5.NELF-AC binds single-stranded nucleic acids.

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