Structural visualization of the p53/RNA polymerase II assembly

Singh, Sameer; Qiao, Zhen; Song, Lihua; Jani, Vijay; Rice, William J.; Eng, Edward T.; Coleman, Robert A.; Liu, Wei Li

doi:10.1101/gad.285692.116

Cited by 14 publications

(29 citation statements)

References 74 publications

(110 reference statements)

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“…S6. We suspect that p53's rapid removal could potentially lead to faster "repurposing" of the RE for additional rounds of p53-mediated recruitment of other basal transcription factors such as TFIIB (55,56), RNA polymerase II (32), and TFIIH (31,52,57). Stable prolonged binding of p53 to a TFIID/DNA complex can potentially negatively regulate PIC formation and squelch transcription.…”

Section: Discussionmentioning

confidence: 99%

“…Consistent with our single-molecule studies, EM structural analysis revealed that conversion of canonical TFIID to a rearranged DNA binding conformation is enhanced in the presence of p53 and DNA. TFIID/DNA binding induces p53 dissociation, which may potentially aid subsequent recruitment of p53 cocomplexes (e.g., p53/Mediator [30], p53/TFIIH [31], and p53/RNA polymerase II [32]) involved in PIC formation. Therefore, proper responses to divergent stress signals involve conserved structural mechanisms relating to p53's ability to dynamically stimulate TFIID's interaction with various target promoters.…”

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confidence: 99%

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p53 Dynamically Directs TFIID Assembly on Target Gene Promoters

Coleman

Qiao

Singh

et al. 2017

Molecular and Cellular Biology

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p53 is a central regulator that turns on vast gene networks to maintain cellular integrity in the presence of various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key issue, we have undertaken an integrated approach involving single-molecule fluorescence microscopy, single-particle cryo-electron microscopy, and biochemistry. Our real-time single-molecule imaging data demonstrate that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID's ability to bind DNA by stabilizing TFIID contacts with both the core promoter and a region within p53's response element. Analysis of single-molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID's conversion to a rearranged DNA binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID's interaction with DNA induces p53 to rapidly dissociate, which likely leads to additional rounds of p53-mediated recruitment of other basal factors. Collectively, these findings indicate that p53 dynamically escorts and loads TFIID onto its target promoters. KEYWORDS p53, TFIID, transcription, structure, single-molecule microscopy M ore than 50% of cancer patients harbor p53 mutations, highlighting the essential role of this protein in tumor suppression (1). To properly maintain genomic stability, p53 induces vast gene networks involved in diverse cellular pathways, including the cell cycle arrest, apoptosis, and DNA repair pathways (2). In response to various stress stimuli, p53 acts as a transcriptional activator that specifically binds consensus response elements (REs; two 10-base-pair half-sites [RRRCWWGYYY]) within its target genes to directly stimulate gene expression (2). p53 utilizes its ability to nonspecifically bind and slide along the DNA to expedite the search for target REs (3). Upon recognition of its response genes, p53 facilitates transcription at least in part by targeting the TFIID-mediated transcription machinery to the promoter (4-7). TFIID is composed of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TFIID recognizes and binds multiple core promoter elements (e.g., the TATA box, the initiator, and downstream core promoter elements [DPE]) surrounding the transcription start site (TSS) (8). Once bound, TFIID serves as a central scaffold for six basal factors, including RNA polymerase II, to form a preinitiation complex (PIC) directing transcription initiation. Importantly, without the assistance of activators such as p53, TFIID's core promoter recognition is weak and rate limiting for transcription initiation due to inefficient PIC assembly (9-16). As TFIID is responsible for transcription of at least ϳ90% of proteincoding genes (17), unraveling how p...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

p53 Dynamically Directs TFIID Assembly on Target Gene Promoters

Coleman

Qiao

Singh

et al. 2017

Molecular and Cellular Biology

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“…While p53 dissociates from the TFIID/DNA scaffold, it is highly likely that p53 will rapidly re-associate with the complex, particularly at the high concentrations (600nM) used in Supplementary Figure S6. We suspect that p53’s rapid removal could consequently lead to a faster “repurposing” of the RE for additional rounds of p53-mediated recruitment of other basal transcription factors such as TFIIB (51, 52), RNA Pol II (53), and TFIIH (49, 54, 55). In this scenario, stable prolonged binding of p53 to a TFIID/DNA complex would negatively regulate PIC formation, because p53 would occupy the RE and inhibit subsequent recruitment of a p53/basal factor co-complex.…”

Section: Discussionmentioning

confidence: 99%

p53 dynamically directs TFIID assembly on target gene promoters

Coleman

Qiao

Singh

et al. 2016

Preprint

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The p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID’s ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53’s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID’s conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID’s interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

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“…Our cryo-EM studies further suggest that p53 aids in TFIID's transition to the rearranged DNA-binding conformation. 7 More recently, armed with significant advancement of cryo-EM, we have captured the first 3D structure of wild-type p53 bound to human Pol II 13 (Fig. 1).…”

Section: Capturing P53-mediated Transcription Assemblies Via Cryo-emmentioning

confidence: 99%

“…6,7 p53 was shown to bind several PIC components (i.e., TFIIB, TFIID, TFIIH, Mediator, Pol II) and subsequently aid their recruitment to the core promoter. [6][7][8][9][10][11][12][13] Thus, the interactions of p53 with multiple components of the PIC are crucial for transcription initiation. Researchers have studied stimulation of eukaryotic transcription by activators (e.g., p53) for the last 30 years.…”

Section: Introductionmentioning

confidence: 99%