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. 2016 Oct 7;11(10):e0163649.
doi: 10.1371/journal.pone.0163649. eCollection 2016.

Gdown1 Associates Efficiently with RNA Polymerase II after Promoter Clearance and Displaces TFIIF during Transcript Elongation

Elizabeth DeLaney  1 , Donal S Luse  1
Affiliations

Gdown1 Associates Efficiently with RNA Polymerase II after Promoter Clearance and Displaces TFIIF during Transcript Elongation

Elizabeth DeLaney et al. PLoS One. .

Abstract

Pausing during the earliest stage of transcript elongation by RNA polymerase II (Pol II) is a nearly universal control point in metazoan gene expression. The substoichiometric Pol II subunit Gdown1 facilitates promoter proximal pausing in vitro in extract-based transcription reactions, out-competes the initiation/elongation factor TFIIF for binding to free Pol II and co-localizes with paused Pol II in vivo. However, we have shown that Gdown1 cannot functionally associate with the Pol II preinitiation complex (PIC), which contains TFIIF. In the present study, we determined at what point after initiation Gdown1 can associate with Pol II and how rapidly this competition with TFIIF occurs. We show that, as with the PIC, Gdown1 cannot functionally load into open complexes or complexes engaged in abortive synthesis of very short RNAs. Gdown1 can load into early elongation complexes (EECs) with 5-9 nt RNAs, but efficient association with EECs does not take place until the point at which the upstream segment of the long initial transcription bubble reanneals. Tests of EECs assembled on a series of promoter variants confirm that this bubble collapse transition, and not transcript length, modulates Gdown1 functional affinity. Gdown1 displaces TFIIF effectively from all complexes downstream of the collapse transition, but this displacement is surprisingly slow: complete loss of TFIIF stimulation of elongation requires 5 min of incubation with Gdown1. The relatively slow functional loading of Gdown1 in the presence of TFIIF suggests that Gdown1 works in promoter-proximal pausing by locking in the paused state after elongation is already antagonized by other factors, including DSIF, NELF and possibly the first downstream nucleosome.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Full functional loading of Gdown1 to EECs takes nearly five minutes.
(A) PICs were assembled with TFIIF containing full-length RAP74 in solution on the AdML 31g(M) template [19] and EECs stalled at +30 were generated with ATP, UTP, and [α-32P]CTP as described in Materials and Methods. EECs were then incubated with 240 fmol of Gdown1 for 0 to 10 min (except lanes 1 and 2) and chased (except lane 1) for 30 sec with 200 μM NTPs. 240 fmol of Gdown1 is a 20-fold excess over Pol II and a 2-fold excess over TFIIF. DNA size markers were run in the far left lane of the gel and their lengths are indicated. Lengths of RNAs in the chase reactions were quantified as TFIIF-dependent and TFIIF-independent as noted on the right. A schematic of the assay is shown to the lower right of the gel. (B) Average percents of TFIIF-dependent transcription were quantified from results like those shown in panel (A) as described in Materials and Methods. The error bars indicate the mean ± S.D. based on five replicates.
Fig 2
Fig 2. Truncation of the large subunit of TFIIF does not enhance functional Gdown1 loading to EECs.
(A) PICs were assembled with 1–227 TFIIF and EECs were generated in solution on the AdML 31g(M) template with ATP, UTP and [α-32P]CTP as described in Materials and Methods. EECs were then incubated with 240 fmol of Gdown1 for 0 to 10 min (except lane 1) and chased for 30 sec with 200 μM NTPs. Lengths of markers are shown on the left edge of the gel, and lengths of TFIIF-dependent and TFIIF-independent transcripts are noted on the right. A schematic of the assay is shown to the lower right of the gel. (B) Average percents of TFIIF-dependent transcription (open bars) were quantified from results like those shown in panel (A) as described in Materials and Methods. The error bars indicate the mean ± S.D. based on four replicates. The grey bars duplicate the values from Fig 1.
Fig 3
Fig 3. Gdown1 is unable to functionally associate with PICs.
PICs were generated on bead bound AdML 31g(M) templates as described in Materials and Methods and then incubated for 5 min with 240 fmol of Gdown1 (lanes 7–8) or buffer (lanes 1–6). The supernatant was removed and replaced with buffer M5 and EECs were generated with pulse labeling as described in Materials and Methods. For lanes 3 and 4, EECs were then incubated with 240 fmol of Gdown1 for 5 min. At this point the reactions were stopped (P lanes) or supplemented with TFIIF and chased (C lanes) for 60 sec with 200 μM NTPs as described in Materials and Methods. Lengths of markers are shown on the left edge of the gel, and TFIIF-dependent and TFIIF-independent transcript lengths are noted on the right. A schematic of the assays in lanes 5–7 is shown to the right of the gel. The gel shown is representative of three replicates that were performed.
Fig 4
Fig 4. Gdown1 does not functionally associate with Pol II in open complex or with initiating Pol II.
(A) PICs on bead bound AdML 31g(M) templates were converted to open complexes by incubation with ATP for 5 min, along with 240 fmole Gdown1 (lanes 3 and 4 only) as described in Materials and Methods. After the supernates were removed and replaced with buffer M5, reactions were supplemented with TFIIE and EECs were generated by pulse labeling with ATP, UTP and [α-32P]CTP as described in Materials and Methods. Reactions were either stopped at this point (P lanes) or supplemented with TFIIF and chased (C lanes) for 60 sec with 200 μM NTPs as described in Materials and Methods. Lengths of markers are shown on the left edge of the gel and TFIIF-dependent and TFIIF-independent transcript lengths are noted on the right. A schematic of the assay is shown to the left of the gel. The gel shown is representative of three replicates that were performed. (B) For lanes 4–7, complexes undergoing abortive initiation were generated on bead bound AdML 31g(M) templates with CpA and CTP as described in Materials and Methods. Reactions in lanes 6 and 7 contained 240 fmol of Gdown1. After 5 minutes the supernatant was removed and replaced with MEMDM40. EECs were then generated with ATP, UTP and [α-32P]CTP as described in Materials and Methods. Reactions were either stopped at this point (P lanes) or supplemented with TFIIF and chased (C lanes) for 60 sec with 200 μM NTPs. Control reactions shown in lanes 1–3 were performed as described in Fig 3 (EEC lanes). The lane 1 reaction was stopped after the pulse (P) step; for the two chase (C) reactions, Gdown1 was incubated for 5 min with the 30-mer complexes only for lane 3. Lengths of markers are shown on the left edge of the gel, and lengths of TFIIF-dependent and TFIIF-independent transcripts are noted on the right. A schematic of the assay in lanes 4–7 is shown to the left of the gel. The gel shown is representative of three replicates that were performed.
Fig 5
Fig 5. Efficiency of Gdown1 inhibition of TFIIF increases significantly with a 10 nt nascent RNA.
(A) EECs were generated in solution on AdML 6g, 8g, 9g, 11g, and 31g(M) [19,30] templates with ATP, UTP and [α-32P]CTP as described in Materials and Methods. EECs were incubated with 240 fmol of Gdown1 or buffer for 5 min as indicated and then chased for 60 sec with 200 μM NTPs. Lengths of markers are shown on the left edge of the gel and TFIIF-dependent and TFIIF-independent transcript lengths are noted on the right. A schematic of the assay is shown to the left of the gel. Spaces in between lanes separate non-adjacent lanes within the same gel. In a separate experiment (gel not shown), reactions were performed as just described but using 21g templates [19] attached to beads to generate complexes with 20 nt RNAs. TFIIF-dependent and independent transcript lengths were determined as for the solution reactions. (B) 20% denaturing PAGE showing the nascent RNAs generated following the initial pulse. (Initial transcripts are not shown for the 21g bead-attached templates.) Arrows on the right indicate the expected transcript size relative to the G stop in the template. The space in between lanes 3 and 4 separates non-adjacent lanes within the same gel. (C) Average percents of TFIIF-dependent transcripts were quantified from results like those shown in panel (A) as described in Materials and Methods. The error bars indicate mean ± S.D. based on three to six replicates per individual template.
Fig 6
Fig 6. Functional binding of Gdown1 to EECs is enhanced following transcription bubble collapse.
(A) Schematic showing the expected sizes of transcription bubbles for EECs paused at the initial G stops on the templates used in this figure (see [30]). (B) 20% denaturing PAGE showing the nascent RNAs generated following the initial pulse. Arrows on the right indicate the expected transcript size relative to the G stop in the template. (C) EECs were generated in solution on AdML 9g, 9g2I, 11g, and 11g2D templates with CpA, dATP, UTP, and [α-32P]CTP as described in Materials and Methods. EECs were then incubated with 240 fmol of Gdown1 or buffer for 5 min and chased for 30 sec with 200 μM NTPs. Lengths of markers are shown on the left edge of the gel and TFIIF-dependent and TFIIF-independent transcript lengths are noted on the right. A schematic of the assay is shown to the left of the gel. (D) Average percents of TFIIF-dependent transcription were quantified from results like those shown in panel (C) as described in Materials and Methods. The error bars indicate mean ± S.D. based on three to six replicates per individual template.
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Grants and funding

This work was supported by grant 1121210 from the National Science Foundation (US). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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