Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex

Abstract

Negative elongation factor (NELF) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) are involved in pausing RNA Polymerase II (Pol II) in the promoter-proximal region of the hsp70 gene in Drosophila, before heat shock induction. Such blocks in elongation are widespread in the Drosophila genome. However, the mechanism by which DSIF and NELF participate in setting up the paused Pol II remains unclear. We analyzed the interactions among DSIF, NELF, and a reconstituted Drosophila Pol II elongation complex to gain insight into the mechanism of pausing. Our results show that DSIF and NELF require a nascent transcript longer than 18 nt to stably associate with the Pol II elongation complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex. Taken together, these results provide a possible model by which DSIF binds the elongation complex via association with the nascent transcript and subsequently recruits NELF. Although DSIF and NELF were both required for inhibition of transcription, we did not detect a NELF-RNA contact when the nascent transcript was between 22 and 31 nt long, which encompasses the region where promoter-proximal pausing occurs on many genes in Drosophila. This raises the possibility that RNA binding by NELF is not necessary in promoter-proximal pausing.

Fig. 1.

Purification of Drosophila NELF. (A) Nuclear extracts were prepared from transgenic fly embryos expressing FLAG-NELF subunits and fractionated using anti-FLAG Sepharose (Sigma). Eluates (10 uL) were analyzed by SDS-PAGE and Coomassie staining (lanes 2–4). Arrows indicate positions of the NELF subunits. Lane 1 (Control) shows 10 uL of eluate from a mock purification of nuclear extract from nontransgenic embryos. Asterisks denote proteins that bind nonspecifically to the FLAG column. (B) Western blot analysis of the FLAG NELF-D complex using NELF-A, NELF-B, NELF-D, and NELF-E antibodies. (C) Silver-stained gel with Pol II purified from Drosophila embryo nuclear extract and Coomassie blue-stained gel with DSIF purified from baculovirus. The prominent band migrating just above Rpb3 is an unidentified contaminant.

Fig. 2.

NELF and DSIF inhibit transcription elongation. (A) Schematic representation of the in vitro elongation system. RNA Pol II initiates transcription with UpG on the tailed template and stalls before the four Gs located at the end of the G-less cassette when GTP is absent. Upon addition of GTP, Pol II is able to resume elongation and generate a runoff transcript. (B) Outline of the experiment to measure the elongation rate of Pol II in the presence or absence of DSIF and NELF. (C) Analysis of elongation rates. Prior to addition of GTP, the stalled elongation complexes were incubated with buffer (lanes 2–5), DSIF (lanes 10–13), NELF (lanes 14–17), or DSIF and NELF (lanes 6–9). Lane 1 shows transcripts isolated from the stalled elongation complex before addition of proteins and GTP.

Fig. 3.

DSIF and NELF form a stable complex with the Pol II elongation complex. (A) Native gel analysis of elongation complexes. Lane 1 shows a complex of Pol II with a 70-nt radiolabeled nascent transcript and the tailed template (EC70). Approximately 100 ng (∼0.25 pmol) of Pol II was present in each sample. Elongation complexes stalled at the end of the G-less cassette were incubated with increasing amounts of purified DSIF (approximately 0.5 pmol, 1 pmol, and 2 pmol of DSIF; lanes 2–4) or purified NELF (approximately 0.15 pmol, 0.3 pmol, and 0.6 pmol of NELF; lanes 5–7). Lanes 8 and 9 show elongation complexes incubated with 0.5 pmol of DSIF and 0.15 pmol of NELF, respectively, and lane 10 shows a complex with both proteins added (0.5 pmol of DSIF and 0.15 pmol of NELF). The concentrations of all proteins were judged by comparing intensities of their bands to that of known amounts of protein markers on a Coomassie-stained gel. Therefore, these concentrations are only estimates and merely indicate that none of the proteins are present in large excess over the others. (B) Evidence for cooperative binding by DSIF and NELF. Approximately 100 ng (∼0.25 pmol) of Pol II was present in each sample. Lane 1 shows EC70 alone. Lane 2–5 have 0.25, 0.5, 0.75, and 1.0 pmol of DSIF and no NELF. Lanes 6–9 have 0.05, 0.1, 0.15, and 0.2 pmol NELF and 0.25 pmol of DSIF. Lanes 10–13 have 0.05, 0.1, 0.15, and 0.2 pmol NELF and 0.5 pmol of DSIF.

Fig. 4.

Binding of DSIF and NELF depends on the length of the RNA. (A) Schematic showing elongation complexes with different lengths of the nascent RNA generated by modifying the length of the G-less cassette. (B) Analysis of RNA produced by transcription on templates containing 70-, 31-, 27-, 22-, 18-, or 14-nt-long G-less cassettes on a denaturing gel. (C) Binding of DSIF and NELF to elongation complexes with different lengths of nascent transcripts. Elongation complexes containing 22-, 18-, or 14-nt-long nascent transcripts were analyzed on a native gel (lanes 1, 5, and 9). Each complex was incubated with 1 pmol of DSIF (lanes 2, 6, and 10) or 0.3 pmol of NELF (lanes 3, 7, and 11) or 1 pmol of DSIF and 0.3 pmol of NELF (lanes 4, 8, and 12).

Fig. 5.

Spt5 contacts the nascent RNA. (A) Results of UV cross-linking of transcription complexes EC31 and EC18. The complexes were left untreated (lanes 2 and 6) or incubated with DSIF and NELF (lanes 4 and 8) before cross-linking. Samples were then treated with RNase A and DNase I, precipitated with trichloroacetic acid, and subjected to SDS-PAGE on a 4%–20% gradient gel (Bio-Rad). Lanes 1, 3, 5, and 7 represent non-cross-linked samples. The band marked with the asterisk may be Rpb7 (see text). (B) Immunoprecipitation of the cross-linked EC31 complex bound to DSIF and NELF. The antibodies used are shown above each lane. The Rpb1 antibody, 8WG16, associates with the CTD of Rpb1. Forty percent of the inputs were loaded in lane 1. The samples were analyzed by 8% SDS-PAGE. The sequence of the nascent transcript in EC31 and EC18 is shown at the bottom. The cross-linkable Us are underlined, and the radioactive Cs are marked with asterisks.

Fig. 6.

NELF-E contacts the RNA in EC70. (A) UV cross-linking of transcription complex EC70 bound to DSIF only (lane 1) or DSIF and NELF (lane 2). (B) Immunoprecipitation of EC70 by different antibodies. (Inset) Part of the gel with enhanced contrast to display the NELF-E band more clearly. Image processing was done using the curves tool in Adobe Photoshop. The sequence of the nascent transcript in EC70 is shown and annotated as described in Fig. 5.