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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

Model of NELF architecture and RNA binding regions.The NELF-AC crystal structure, crosslinking data, and in vivo experiments reveal the overall architecture of NELF. NELF-AC (NELF-A red, NELF-C cyan) forms a stable complex that interacts with the N-terminus of NELF-B (orange). NELF-B N- and C- termini sandwich the NELF-E N-terminus. The NELF-E RRM is loosely connected to the entire complex. RNA binds to NELF-E, NELF-B, and NELF-C. The two faces of NELF-AC determined by crystallography and crosslinking are shown below the complex in cartoon format. Three RNA binding faces on the surface of NELF-C are marked with numbers (1, 2, 4). The region of NELF-A that is predicted to bind Pol II, which is absent in our crystal structure is boxed. N- and C- termini for each protein are marked.DOI:http://dx.doi.org/10.7554/eLife.14981.024
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fig9: Model of NELF architecture and RNA binding regions.The NELF-AC crystal structure, crosslinking data, and in vivo experiments reveal the overall architecture of NELF. NELF-AC (NELF-A red, NELF-C cyan) forms a stable complex that interacts with the N-terminus of NELF-B (orange). NELF-B N- and C- termini sandwich the NELF-E N-terminus. The NELF-E RRM is loosely connected to the entire complex. RNA binds to NELF-E, NELF-B, and NELF-C. The two faces of NELF-AC determined by crystallography and crosslinking are shown below the complex in cartoon format. Three RNA binding faces on the surface of NELF-C are marked with numbers (1, 2, 4). The region of NELF-A that is predicted to bind Pol II, which is absent in our crystal structure is boxed. N- and C- termini for each protein are marked.DOI:http://dx.doi.org/10.7554/eLife.14981.024

Mentions: From our structural, biochemical and in vivo data, we propose an architectural model for the NELF complex. In the complex, NELF-AC binds to the N-terminal region of NELF-B. The N-terminal region of NELF-E is sandwiched in between the N- and C- termini of NELF-B, anchoring the flexible NELF-E RRM domain to the rest of the complex. Strong RNA binding by the NELF-E RRM may initially recruit NELF to RNA and secondary binding events by NELF-B and NELF-C may further stabilize the complex on nucleic acid. Future structural studies are required to determine the nucleic acid binding surfaces of NELF-B and to determine how RNA snakes from the NELF-E RRM through the rest of the complex (Figure 9). It is also likely that RNA-binding involves major conformational changes.10.7554/eLife.14981.024Figure 9.Model of NELF architecture and RNA binding regions.


Architecture and RNA binding of the human negative elongation factor
Model of NELF architecture and RNA binding regions.The NELF-AC crystal structure, crosslinking data, and in vivo experiments reveal the overall architecture of NELF. NELF-AC (NELF-A red, NELF-C cyan) forms a stable complex that interacts with the N-terminus of NELF-B (orange). NELF-B N- and C- termini sandwich the NELF-E N-terminus. The NELF-E RRM is loosely connected to the entire complex. RNA binds to NELF-E, NELF-B, and NELF-C. The two faces of NELF-AC determined by crystallography and crosslinking are shown below the complex in cartoon format. Three RNA binding faces on the surface of NELF-C are marked with numbers (1, 2, 4). The region of NELF-A that is predicted to bind Pol II, which is absent in our crystal structure is boxed. N- and C- termini for each protein are marked.DOI:http://dx.doi.org/10.7554/eLife.14981.024
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Related In: Results  -  Collection

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fig9: Model of NELF architecture and RNA binding regions.The NELF-AC crystal structure, crosslinking data, and in vivo experiments reveal the overall architecture of NELF. NELF-AC (NELF-A red, NELF-C cyan) forms a stable complex that interacts with the N-terminus of NELF-B (orange). NELF-B N- and C- termini sandwich the NELF-E N-terminus. The NELF-E RRM is loosely connected to the entire complex. RNA binds to NELF-E, NELF-B, and NELF-C. The two faces of NELF-AC determined by crystallography and crosslinking are shown below the complex in cartoon format. Three RNA binding faces on the surface of NELF-C are marked with numbers (1, 2, 4). The region of NELF-A that is predicted to bind Pol II, which is absent in our crystal structure is boxed. N- and C- termini for each protein are marked.DOI:http://dx.doi.org/10.7554/eLife.14981.024
Mentions: From our structural, biochemical and in vivo data, we propose an architectural model for the NELF complex. In the complex, NELF-AC binds to the N-terminal region of NELF-B. The N-terminal region of NELF-E is sandwiched in between the N- and C- termini of NELF-B, anchoring the flexible NELF-E RRM domain to the rest of the complex. Strong RNA binding by the NELF-E RRM may initially recruit NELF to RNA and secondary binding events by NELF-B and NELF-C may further stabilize the complex on nucleic acid. Future structural studies are required to determine the nucleic acid binding surfaces of NELF-B and to determine how RNA snakes from the NELF-E RRM through the rest of the complex (Figure 9). It is also likely that RNA-binding involves major conformational changes.10.7554/eLife.14981.024Figure 9.Model of NELF architecture and RNA binding regions.

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