A single-molecule characterization of p53 search on DNA

Tafvizi, Anahita; Huang, Fang; Fersht, Alan R.; Mirny, Leonid A.; Oijen, Antoine M. van

doi:10.1073/pnas.1016020107

Cited by 222 publications

(329 citation statements)

References 36 publications

(54 reference statements)

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“…Acetylation of Lys120 is now seen to increase the specificity of p53 and change its conformational preferences. If the data in vitro extend to the situation in vivo, then p53 prior to acetylation of Lys120 would not be in a search process for its targets, but would bind to any exposed nonspecific sequences, possibly sliding along them (35). Acetylation would then start the search process by weakening the binding to nonspecific sequences.…”

Section: Binomial Distribution Experimentalmentioning

confidence: 99%

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Arbely

Natan

Brandt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-Å resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNAðp53DBDÞ 4 or DNA ðAcK120DBDÞ 4 , indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.

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Section: Binomial Distribution Experimentalmentioning

confidence: 99%

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Arbely

Natan

Brandt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…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)(5)(6)(7).…”

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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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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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“…Recent single-molecule experiments revealed that the diffusion coefficients for 1D sliding along DNA are two or more orders of magnitude lower than the 3D diffusion coefficients [20,21,[51][52][53]. Previously it was often assumed that for reaction acceleration by reduction of dimensionality to occur, the 1D diffusion coefficients have to be higher or of the same order of magnitude as the related 3D diffusion coefficients [54].…”

Section: Numerical Simulation and Discussionmentioning

confidence: 99%

Theory of bimolecular reactions in a solution with linear traps: Application to the problem of target search on DNA

2015

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Turkin, A. (2015). Theory of bimolecular reactions in a solution with linear traps: application to the problem of target search on DNA. Physical Review E: Statistical, Nonlinear, and Soft Matter Physics, 92 (5), 52703-1 -52703-14.Theory of bimolecular reactions in a solution with linear traps: application to the problem of target search on DNA AbstractOne-dimensional sliding along DNA as a means to accelerate protein target search is a well-known phenomenon occurring in various biological systems. Using a biomimetic approach, we have recently demonstrated the practical use of DNA-sliding peptides to speed up bimolecular reactions more than an order of magnitude by allowing the reactants to associate not only in the solution by three-dimensional (3D) diffusion, but also on DNA via one-dimensional (1D) diffusion [A. Turkin et al., Chem. Sci. (2015)]. Here we present a mean-field kinetic model of a bimolecular reaction in a solution with linear extended sinks (e.g., DNA) that can intermittently trap molecules present in a solution. The model consists of chemical rate equations for mean concentrations of reacting species. Our model demonstrates that addition of linear traps to the solution can significantly accelerate reactant association. We show that at optimum concentrations of linear traps the 1D reaction pathway dominates in the kinetics of the bimolecular reaction; i.e., these 1D traps function as an assembly line of the reaction product. Moreover, we show that the association reaction on linear sinks between trapped reactants exhibits a nonclassical third-order behavior. Predictions of the model agree well with our experimental observations. Our model provides a general description of bimolecular reactions that are controlled by a combined 3D+1D mechanism and can be used to quantitatively describe both naturally occurring as well as biomimetic biochemical systems that reduce the dimensionality of search. Theory of bimolecular reactions in a solution with linear traps:Application to the problem of target search on DNA One-dimensional sliding along DNA as a means to accelerate protein target search is a well-known phenomenon occurring in various biological systems. Using a biomimetic approach, we have recently demonstrated the practical use of DNA-sliding peptides to speed up bimolecular reactions more than an order of magnitude by allowing the reactants to associate not only in the solution by three-dimensional (3D) diffusion, but also on DNA via one-dimensional (1D) diffusion [A. Turkin et al., Chem. Sci. (2015)]. Here we present a mean-field kinetic model of a bimolecular reaction in a solution with linear extended sinks (e.g., DNA) that can intermittently trap molecules present in a solution. The model consists of chemical rate equations for mean concentrations of reacting species. Our model demonstrates that addition of linear traps to the solution can significantly accelerate reactant association. We show that at optimum concentrations of linear traps the 1D reaction pathway dominates in the kinetics of...

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A single-molecule characterization of p53 search on DNA

Cited by 222 publications

References 36 publications

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

p53 Dynamically Directs TFIID Assembly on Target Gene Promoters

Theory of bimolecular reactions in a solution with linear traps: Application to the problem of target search on DNA

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