Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization

Ly, Elina; Goodrich, James A.; Kugel, Jennifer F.

doi:10.1016/j.ymeth.2019.03.006

Cited by 9 publications

(9 citation statements)

References 16 publications

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“…Cleaning and assembly of the flow chambers and preparation of stock solutions of streptavidin, d -glucose, glucose oxidase, catalase, and 100 mM Trolox (Sigma) for single molecule microscopy were as previously described 52 . All single molecule binding assays were performed at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

Kugel

Goodrich

2020

Sci Rep

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The tumor suppressor protein p53 is critical for cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds DNA response elements to regulate transcription of target genes. p53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding DNA, unanswered questions remain, due partly to the need for more quantitative and structural studies with full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding DNA in real time. The data revealed a dynamic interaction in which tetrameric p53/DNA complexes assembled and disassembled without a dimer/DNA intermediate. On a wild type DNA containing two half sites, p53/DNA complexes existed in two kinetically distinct populations. p53 tetramers bound response elements containing only one half site to form a single population of complexes with reduced kinetic stability. Altering the spacing and helical phasing between two half sites affected both the population distribution of p53/DNA complexes and their kinetic stability. Our real time single molecule measurements of full length p53 tetramers binding DNA reveal the parameters that define the stability of p53/DNA complexes, and provide insight into the pathways by which those complexes assemble.

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Section: Methodsmentioning

confidence: 99%

Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

Kugel

Goodrich

2020

Sci Rep

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“…The cleaning and assembly of the flow chambers, the surface functionalization, and the preparation of streptavidin, D-glucose, glucose oxidase, catalase, and 100 mM Trolox stock solutions were as previously described [30]. Preinitiation complexes were formed by incubating 0.15-0.25 nM biotinylated three piece DNA with 3.5 nM TBP, 5 nM AF647-TFIIB, 2 nM TFIIF, (which was not certified by peer review) is the author/funder.…”

Section: Single Molecule Transcription Assaysmentioning

confidence: 99%

Release of Human TFIIB from Actively Transcribing Complexes Is Triggered upon Synthesis of 7- and 9-nt RNAs

Chen

Goodrich

et al. 2020

Journal of Molecular Biology

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RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription.Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important to maintain the transcriptional activity of Pol II as initiating complexes transition into elongation complexes.

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“…To further investigate the direct transfer model, we employed total internal reflection fluorescence (TIRF) microscopy-based single-molecule biophysics (SMB) experiments (33) as an orthogonal approach to rule out FP-dependent artifacts. We chose the TREX1 + 5-mer ssDNA interaction for these experiments because it was well behaved.…”

Section: Resultsmentioning

confidence: 99%

“…PEG-biotin coated microscope slides were prepared as previously described (33). Slides were (1) washed twice with 200 µL Millipore water (mpH2O), (2) twice with 200 µL reaction buffer (BB2 + 0.05% v/v NP-40), (3) incubated for 5 min with a mix of 0.2 mg/mL streptavidin and 0.8 mg/mL BSA in reaction buffer, (4) washed twice with 200 µL reaction buffer, (5) incubated for 5 min with 25 pM (single-label) or 5 pM (dual-label) biotin-tagged α-FLAG monoclonal antibody (ThermoFisher Scientific #MA1-91878-BTIN, Lot #XB341977) in reaction buffer, (6) washed twice with 200 µL reaction buffer, (7) incubated for 5 min with 250 nM TREX1-FLAG protein in reaction buffer, and (8) washed twice with 200 µL reaction buffer.…”

Section: Methodsmentioning

confidence: 99%

RNA- and DNA-binding proteins generally exhibit direct transfer of polynucleotides: Implications for target site search

Hemphill

Voong

Fenske

et al. 2022

Preprint

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We previously demonstrated that the PRC2 chromatin-modifying enzyme exhibits the ability to directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1 (TREX1), heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally interrogated by single molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated ligands. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, presumably long-lived protein-polynucleotide complexes might instead be readily replaced by other protein-polynucleotide complexesin vivo.SignificanceClassically, the lifetime of a protein-ligand complex is presumed to be an intrinsic property, unaffected by competitor molecules in free solution. By contrast, a few oligomeric nucleic acid binding proteins have been observed to exchange competing ligands in their binding sites, and consequently their lifetimes decrease with competitor concentration. Our findings indicate that this “direct transfer” is a more general property of nucleic acid binding proteins. This suggests that many DNA- and RNA-binding proteins can reduce the dimensionality of their search for their target sites by intramolecular direct transfer to nucleosome DNA, instead of relying entirely on three-dimensional diffusion, and it suggests that their mean complex lifetimesin vivocan be regulated by the concentration of free ligand molecules.

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Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization

Cited by 9 publications

References 16 publications

Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

Release of Human TFIIB from Actively Transcribing Complexes Is Triggered upon Synthesis of 7- and 9-nt RNAs

RNA- and DNA-binding proteins generally exhibit direct transfer of polynucleotides: Implications for target site search

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