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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

Additional information supporting NELF architecture as determined by crosslinking MS.(A) Map of all crosslinks detected between NELF subunits. Crosslinks emanating from NELF-A are red, NELF-C are cyan, and NELF-E are purple. (B) Representative spectra of NELF inter and intraprotein crosslinks as determined by mass spectrometry. Sequences for crosslinked peptides are shown above the spectra. (C) Distances between inter- and intraprotein crosslinks within the NELF-AC crystal structure. Distances were measured in PyMol (PyMOL, 2002). Crosslinks greater than 30Å can be attributed to a conformation not present in the crystal structure between NELF-C helices 12’ and 13’ α helices 18’ and 19’. (D) Model of NELF-B as generated by I-TASSER. The model suggests NELF-B is highly alpha helical in nature and composed of heat repeats. NELF-B lysines that form inter- and intrasubunit crosslinks are indicated on the model. (E) Intrasubunit crosslinks between lysines 260 and 332 of the NELF-E RRM are marked on the NMR structure indicating the RRM fold is maintained in context of the NELF tetramer (PDB ID: 2JX2) (Rao et al., 2006).DOI:http://dx.doi.org/10.7554/eLife.14981.011
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fig3s1: Additional information supporting NELF architecture as determined by crosslinking MS.(A) Map of all crosslinks detected between NELF subunits. Crosslinks emanating from NELF-A are red, NELF-C are cyan, and NELF-E are purple. (B) Representative spectra of NELF inter and intraprotein crosslinks as determined by mass spectrometry. Sequences for crosslinked peptides are shown above the spectra. (C) Distances between inter- and intraprotein crosslinks within the NELF-AC crystal structure. Distances were measured in PyMol (PyMOL, 2002). Crosslinks greater than 30Å can be attributed to a conformation not present in the crystal structure between NELF-C helices 12’ and 13’ α helices 18’ and 19’. (D) Model of NELF-B as generated by I-TASSER. The model suggests NELF-B is highly alpha helical in nature and composed of heat repeats. NELF-B lysines that form inter- and intrasubunit crosslinks are indicated on the model. (E) Intrasubunit crosslinks between lysines 260 and 332 of the NELF-E RRM are marked on the NMR structure indicating the RRM fold is maintained in context of the NELF tetramer (PDB ID: 2JX2) (Rao et al., 2006).DOI:http://dx.doi.org/10.7554/eLife.14981.011

Mentions: To place the NELF-AC crystal structure in context of the NELF tetramer, we determined the architecture of the four-subunit NELF complex by lysine specific crosslinking followed by mass spectrometry. We expressed the full-length four-subunit NELF complex recombinantly in insect cells from a single virus and purified it to homogeneity (Materials and methods, Figure 3A). The purified complex was crosslinked with disuccinimidyl suberate (DSS) and lysine-lysine crosslinks were detected by mass spectrometry as previously described (Herzog et al., 2012). We obtained a total of 424 unique high-confidence lysine-lysine crosslinks, including 279 intersubunit and 145 intrasubunit crosslinks (Figure 3—figure supplement 1A,B, Figure 3—source data 1).10.7554/eLife.14981.009Figure 3.Architecture of human NELF complex as detected by crosslinking MS.


Architecture and RNA binding of the human negative elongation factor
Additional information supporting NELF architecture as determined by crosslinking MS.(A) Map of all crosslinks detected between NELF subunits. Crosslinks emanating from NELF-A are red, NELF-C are cyan, and NELF-E are purple. (B) Representative spectra of NELF inter and intraprotein crosslinks as determined by mass spectrometry. Sequences for crosslinked peptides are shown above the spectra. (C) Distances between inter- and intraprotein crosslinks within the NELF-AC crystal structure. Distances were measured in PyMol (PyMOL, 2002). Crosslinks greater than 30Å can be attributed to a conformation not present in the crystal structure between NELF-C helices 12’ and 13’ α helices 18’ and 19’. (D) Model of NELF-B as generated by I-TASSER. The model suggests NELF-B is highly alpha helical in nature and composed of heat repeats. NELF-B lysines that form inter- and intrasubunit crosslinks are indicated on the model. (E) Intrasubunit crosslinks between lysines 260 and 332 of the NELF-E RRM are marked on the NMR structure indicating the RRM fold is maintained in context of the NELF tetramer (PDB ID: 2JX2) (Rao et al., 2006).DOI:http://dx.doi.org/10.7554/eLife.14981.011
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fig3s1: Additional information supporting NELF architecture as determined by crosslinking MS.(A) Map of all crosslinks detected between NELF subunits. Crosslinks emanating from NELF-A are red, NELF-C are cyan, and NELF-E are purple. (B) Representative spectra of NELF inter and intraprotein crosslinks as determined by mass spectrometry. Sequences for crosslinked peptides are shown above the spectra. (C) Distances between inter- and intraprotein crosslinks within the NELF-AC crystal structure. Distances were measured in PyMol (PyMOL, 2002). Crosslinks greater than 30Å can be attributed to a conformation not present in the crystal structure between NELF-C helices 12’ and 13’ α helices 18’ and 19’. (D) Model of NELF-B as generated by I-TASSER. The model suggests NELF-B is highly alpha helical in nature and composed of heat repeats. NELF-B lysines that form inter- and intrasubunit crosslinks are indicated on the model. (E) Intrasubunit crosslinks between lysines 260 and 332 of the NELF-E RRM are marked on the NMR structure indicating the RRM fold is maintained in context of the NELF tetramer (PDB ID: 2JX2) (Rao et al., 2006).DOI:http://dx.doi.org/10.7554/eLife.14981.011
Mentions: To place the NELF-AC crystal structure in context of the NELF tetramer, we determined the architecture of the four-subunit NELF complex by lysine specific crosslinking followed by mass spectrometry. We expressed the full-length four-subunit NELF complex recombinantly in insect cells from a single virus and purified it to homogeneity (Materials and methods, Figure 3A). The purified complex was crosslinked with disuccinimidyl suberate (DSS) and lysine-lysine crosslinks were detected by mass spectrometry as previously described (Herzog et al., 2012). We obtained a total of 424 unique high-confidence lysine-lysine crosslinks, including 279 intersubunit and 145 intrasubunit crosslinks (Figure 3—figure supplement 1A,B, Figure 3—source data 1).10.7554/eLife.14981.009Figure 3.Architecture of human NELF complex as detected by crosslinking MS.

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