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

Elizabeth Delaney,Donal S Luse,Xuhui Huang

doi:10.1371/journal.pone.0163649

Elizabeth Delaney, Donal S Luse + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0163649

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Journal: PloS one	Publication Date: Oct 7, 2016
Citations: 17	License type: CC BY 4.0

Affiliation: Cleveland Clinic Lerner College of Medicine

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.

Highlights

The promoter proximal pause, in which Pol II typically accumulates at about 50 bp downstream of transcription start, is a universal feature of transcription by RNA polymerase II in metazoans
By challenging these early elongation complexes (EECs) with Gdown1 followed by chase with all four NTPs, we can assess the extent to which Gdown1 has converted the complexes from rapidly elongating to slowly elongating
This approach exploits the fact that TFIIF cannot displace Gdown1 once Gdown1 has bound to Pol II [9]

Summary

Introduction

The promoter proximal pause, in which Pol II typically accumulates at about 50 bp downstream of transcription start, is a universal feature of transcription by RNA polymerase II in metazoans. Promoter proximal pausing plays a critical regulatory role in transcription and disruption of pausing can have widespread deleterious effects on gene expression [1,2,3]. The best approximation of the +50 pause in vitro has been observed using transcription with nuclear extracts. Those studies have identified Gdown as an important additional component in establishing the promoter proximal pause by Pol II [6]. When Gdown was added to extract-based transcription reactions in which escape into productive elongation had been blocked, Gdown strongly increased pausing, resulting in promoter proximal accumulation of Pol II [6]. ChIP assays demonstrated that Gdown co-localizes with Pol II in the promoter proximal region in vivo; the ratio of Gdown to Pol II was found to be highest at poorly expressed genes [6]

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Conclusion