Fig 2: 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

Fig 3: Surface properties of NELF-AC.Two views of the solvent-accessible surface related by 180° rotations around a vertical axis are shown. (A) Surface representation of NELF-AC with shaded ellipses representing regions where NELF-B (orange) or NELF-E (purple) crosslinks with NELF-AC were detected. Lysines are shown in stick representation and numbered. NELF-B and NELF-E primarily crosslink to one face of the NELF-AC dimer. Four empty ellipses represent patches of positively charged residues. (B) Surface conservation. Residues that are invariant from human to Drosophila are in green, conserved residues in yellow (Figure 1B). Surface areas involved in nucleic acid binding (patches 1–4) (Results) are highlighted. Colors of labels according to color code of protein features belong to (Figure 2). (C) Electrostatic surface potential generated with ABPS (Baker et al., 2001). Blue, red, and white areas indicate positive, negative and neutral charge, respectively. Surface areas involved in nucleic acid binding (patches 1–4). Colors of labels according to color code of protein features belong to (Figure 2).DOI: http://dx.doi.org/10.7554/eLife.14981.012

Fig 4: NELF association with RNA in cells.(A) Immunoprecipitation of the entire NELF complex by a NELF-E specific antibody from Jurkat cells treated with 4-thiouridine (4sU) for 16 hr and UV-crosslinked as detected by Western blot anaylsis with subunit specific antibodies. Cells treated with 4sU, but not UV-crosslinked are shown as a control. Star indicates NELF-C detected on the same blot. Western blot detected with HRP-conjugated secondary antibodies. (B) Phosphorimage of SDS-PAGE used to resolve RNAs crosslinked to NELF subunits from Panel A. Protein crosslinked to RNA was treated with PNK in the presence of ATP [γ-32P]. The resulting protein was run on a 4–12% gradient SDS-PAGE. The gel was incubated with a phosphorimage screen and imaged with a Typhoon imager. In crosslinked cells, a band is seen for the NELF-E and NELF-B/C subunits. Cells treated with 4sU, but not UV-crosslinked are shown as a control. A Coomassie stained gel with purified NELF is shown as size reference. (C) Phosphorimage of SDS-PAGE used to resolve 5’ P-32 labeled RNAs crosslinked to overexpressed -C (left), or -B (right) 293FT cells. Overexpressed NELF-C/-B were immunoprecipitated by N-terminal 3X FLAG tags. Coomassie stained gels of overexpressed NELF-C and -B are shown as a size reference. See Figure 8—figure supplement 3 for further controls.DOI: http://dx.doi.org/10.7554/eLife.14981.020

Fig 5: Architecture of human NELF complex as detected by crosslinking MS.(A) The four-subunit human NELF complex was recombinantly expressed in insect cells and purified to homogeneity. The purified complex (0.9 µg) was run on a 4–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. A molecular weight marker (MW) in kilodaltons (kDa) is provided on the left side of the gel. (B) Cartoon depiction of individual NELF proteins. Different shades designate unique regions or domains of each protein (NELF-A red, NELF-B orange, NELF-C cyan, NELF-E purple). N- and C-term refers to the N- or C-terminus of a protein, respectively. RD repeats refer to a flexible region of NELF-E that is primarily composed of Arg and Asp residues. (C–H) Crosslinks detected within the NELF tetramer displayed as binary interactions. Intraprotein crosslinks are shown as curves and colored as in (B). Interprotein crosslinks are shown as black lines. The endpoint of each line specifies a specific residue in the corresponding protein. Crosslinking data is filtered to display crosslinks with an ID-score greater than 30. A full map of all interprotein crosslinks is provided in Figure 3—figure supplement 1A. All crosslinks can be found in Figure 3—source data 1. Crosslinking data modeled with xiNET (Combe et al., 2015). (C) NELF-A, NELF-C (D) NELF-B, NELF-A (E) NELF-B, NELF-C (F) NELF-B, NELF-E (G) NELF-E, NELF-C (H) NELF-E, NELF-A.DOI: http://dx.doi.org/10.7554/eLife.14981.00910.7554/eLife.14981.010Figure 3—source data 1.Crosslinking MS of four-subunit NELF complex primary data for intra and interprotein crosslinks.DOI: http://dx.doi.org/10.7554/eLife.14981.010