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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.


Multiple sequence alignment of full-length NELF-A demonstrating the comparatively high conservation of the crystallized region.The alignment compares full-length NELF-A from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker blue representing higher conservation. The dark boxes indicate species-specific sequences that were removed due to repetitiveness and lack of sequence conservation. Barrels above the alignment represent α-helices, and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.14981.004
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fig1s2: Multiple sequence alignment of full-length NELF-A demonstrating the comparatively high conservation of the crystallized region.The alignment compares full-length NELF-A from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker blue representing higher conservation. The dark boxes indicate species-specific sequences that were removed due to repetitiveness and lack of sequence conservation. Barrels above the alignment represent α-helices, and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.14981.004

Mentions: The crystallized regions of human NELF-AC share considerable homology among metazoans, particularly at residues forming the hydrophobic cores and the interface between NELF-A and NELF-C (Figure 1B). The extent of conservation is evident when human and Drosophila melanogaster are compared, which share 55% identity for NELF-A and 50% identity for NELF-C. Intriguingly, NELF-A and -C homologs are present in some worms such as the filiarial nematode Loa loa (Figure 1—figure supplements 2, 3) and single celled organisms such as the green algae Chlorella variabilis and the slime mold Dictyostelium discoideum (Figure 1—figure supplements 2, 3). Most regions outside of the crystallized NELF-AC core diverge between single celled organisms and metazoans (Figure 1—figure supplements 2, 3). Such conservation suggests that NELF may have been present in early eukaryotes and was lost in certain lineages over time.


Architecture and RNA binding of the human negative elongation factor
Multiple sequence alignment of full-length NELF-A demonstrating the comparatively high conservation of the crystallized region.The alignment compares full-length NELF-A from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker blue representing higher conservation. The dark boxes indicate species-specific sequences that were removed due to repetitiveness and lack of sequence conservation. Barrels above the alignment represent α-helices, and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.14981.004
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fig1s2: Multiple sequence alignment of full-length NELF-A demonstrating the comparatively high conservation of the crystallized region.The alignment compares full-length NELF-A from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker blue representing higher conservation. The dark boxes indicate species-specific sequences that were removed due to repetitiveness and lack of sequence conservation. Barrels above the alignment represent α-helices, and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.14981.004
Mentions: The crystallized regions of human NELF-AC share considerable homology among metazoans, particularly at residues forming the hydrophobic cores and the interface between NELF-A and NELF-C (Figure 1B). The extent of conservation is evident when human and Drosophila melanogaster are compared, which share 55% identity for NELF-A and 50% identity for NELF-C. Intriguingly, NELF-A and -C homologs are present in some worms such as the filiarial nematode Loa loa (Figure 1—figure supplements 2, 3) and single celled organisms such as the green algae Chlorella variabilis and the slime mold Dictyostelium discoideum (Figure 1—figure supplements 2, 3). Most regions outside of the crystallized NELF-AC core diverge between single celled organisms and metazoans (Figure 1—figure supplements 2, 3). Such conservation suggests that NELF may have been present in early eukaryotes and was lost in certain lineages over time.

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.