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1.
Annu Rev Biophys ; 2023 Dec 22.
Article in English | MEDLINE | ID: mdl-38134333

ABSTRACT

The translation of messenger RNA (mRNA) into proteins represents the culmination of gene expression. Recent technological advances have revolutionized our ability to investigate this process with unprecedented precision, enabling the study of translation at the single-molecule level in real time within live cells. In this review, we provide an overview of single-mRNA translation reporters. We focus on the core technology, as well as the rapid development of complementary probes, tags, and accessories that enable the visualization and quantification of a wide array of translation dynamics. We then highlight notable studies that have utilized these reporters in model systems to address key biological questions. The high spatiotemporal resolution of these studies is shedding light on previously unseen phenomena, uncovering the full heterogeneity and complexity of translational regulation. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

2.
Sci Adv ; 9(40): eadh4819, 2023 10 06.
Article in English | MEDLINE | ID: mdl-37792937

ABSTRACT

Histone acetylation and RNA polymerase II phosphorylation are associated with transcriptionally active chromatin, but their spatiotemporal relationship in live cells remains poorly understood. To address this problem, we combine Fab-based labeling of endogenous protein modifications with single-molecule tracking to quantify the dynamics of chromatin enriched with histone H3 lysine-27 acetylation (H3K27ac) and RNA polymerase II serine-5 phosphorylation (RNAP2-Ser5ph). Our analysis reveals that chromatin enriched with these two modifications is generally separate. In these separated sites, we show that the two modifications are inversely correlated with one another on the minutes time scale and that single nucleosomes within each region display distinct and opposing dynamics on the subsecond time scale. While nucleosomes diffuse ~15% faster in chromatin enriched with H3K27ac, they diffuse ~15% slower in chromatin enriched with RNAP2-Ser5ph. These results argue that high levels of H3K27ac and RNAP2-Ser5ph are not often present together at the same place and time, but rather each marks distinct transcriptionally poised or active sites, respectively.


Subject(s)
Histones , Nucleosomes , Histones/metabolism , RNA Polymerase II/metabolism , Acetylation , Chromatin/genetics
3.
Front Cell Dev Biol ; 11: 1151318, 2023.
Article in English | MEDLINE | ID: mdl-37325568

ABSTRACT

mRNA translation is the ubiquitous cellular process of reading messenger-RNA strands into functional proteins. Over the past decade, large strides in microscopy techniques have allowed observation of mRNA translation at a single-molecule resolution for self-consistent time-series measurements in live cells. Dubbed Nascent chain tracking (NCT), these methods have explored many temporal dynamics in mRNA translation uncaptured by other experimental methods such as ribosomal profiling, smFISH, pSILAC, BONCAT, or FUNCAT-PLA. However, NCT is currently restricted to the observation of one or two mRNA species at a time due to limits in the number of resolvable fluorescent tags. In this work, we propose a hybrid computational pipeline, where detailed mechanistic simulations produce realistic NCT videos, and machine learning is used to assess potential experimental designs for their ability to resolve multiple mRNA species using a single fluorescent color for all species. Our simulation results show that with careful application this hybrid design strategy could in principle be used to extend the number of mRNA species that could be watched simultaneously within the same cell. We present a simulated example NCT experiment with seven different mRNA species within the same simulated cell and use our ML labeling to identify these spots with 90% accuracy using only two distinct fluorescent tags. We conclude that the proposed extension to the NCT color palette should allow experimentalists to access a plethora of new experimental design possibilities, especially for cell Signaling applications requiring simultaneous study of multiple mRNAs.

4.
bioRxiv ; 2023 Jan 26.
Article in English | MEDLINE | ID: mdl-36747627

ABSTRACT

mRNA translation is the ubiquitous cellular process of reading messenger-RNA strands into functional proteins. Over the past decade, large strides in microscopy techniques have allowed observation of mRNA translation at a single-molecule resolution for self-consistent time-series measurements in live cells. Dubbed Nascent chain tracking (NCT), these methods have explored many temporal dynamics in mRNA translation uncaptured by other experimental methods such as ribosomal profiling, smFISH, pSILAC, BONCAT, or FUNCAT-PLA. However, NCT is currently restricted to the observation of one or two mRNA species at a time due to limits in the number of resolvable fluorescent tags. In this work, we propose a hybrid computational pipeline, where detailed mechanistic simulations produce realistic NCT videos, and machine learning is used to assess potential experimental designs for their ability to resolve multiple mRNA species using a single fluorescent color for all species. Through simulation, we show that with careful application, this hybrid design strategy could in principle be used to extend the number of mRNA species that could be watched simultaneously within the same cell. We present a simulated example NCT experiment with seven different mRNA species within the same simulated cell and use our ML labeling to identify these spots with 90% accuracy using only two distinct fluorescent tags. The proposed extension to the NCT color palette should allow experimentalists to access a plethora of new experimental design possibilities, especially for cell signalling applications requiring simultaneous study of multiple mRNAs.

5.
Nat Commun ; 13(1): 3345, 2022 06 10.
Article in English | MEDLINE | ID: mdl-35688806

ABSTRACT

A major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells. We show that our combined translation and Ago2 tethering sensor reflects endogenous miRNA-mediated gene silencing. Using the sensor, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. Silencing was occasionally interrupted by brief bursts of translational activity and took 3-4 times longer than a single round of translation, consistent with a gradual increase in the inhibition of translation initiation. At later time points, Ago2-tethered mRNAs cluster and coalesce with P-bodies, where a translationally silent state is maintained. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single-molecule level. Furthermore, our tethering-based, single-molecule reporter system will likely have wide-ranging application in studying RNA-protein interactions.


Subject(s)
Argonaute Proteins , MicroRNAs , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Gene Expression Regulation , Gene Silencing , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
6.
Methods Mol Biol ; 2428: 349-360, 2022.
Article in English | MEDLINE | ID: mdl-35171490

ABSTRACT

Single-molecule imaging in living cells enables the investigation of molecular dynamics and interactions underlying the physiology of a cell. We recently developed a method to visualize translation events at single-mRNA resolution in living cells. Here we describe how we apply this method to visualize mRNA interactions with stress granules in the context of translational activity during cell stress.


Subject(s)
Single Molecule Imaging , Stress Granules , Cytoplasmic Granules/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Single Molecule Imaging/methods
7.
Bio Protoc ; 11(14): e4096, 2021 Jul 20.
Article in English | MEDLINE | ID: mdl-34395732

ABSTRACT

Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind complementary epitopes in the two separate ORFs of the bicistronic biosensor. This causes the biosensor to fluoresce in different colors depending on which ORF/epitopes are translated. Using the biosensor together with high-resolution fluorescence microscopy and single-molecule tracking analysis allows for the quantitative comparison of translation dynamics between the two ORFs at a resolution of tens-of-nanometers in space and sub-seconds in time, which is not possible with more traditional GFP or luciferase reporters. Since both ORFs are on the same biosensor, they experience the same microenvironment, allowing a fair comparison of their relative translational activities. In this protocol, we describe how to get this assay up and running in cultured human cells so that translation dynamics can be studied under both normal and stressful cellular conditions. We also provide a number of useful tips and notes to help express components at appropriate levels inside cells for optimal live cell imaging. Graphical abstract: Steps required for 3-color single-molecule translation imaging and analysis.

8.
Nat Commun ; 12(1): 3158, 2021 05 26.
Article in English | MEDLINE | ID: mdl-34039974

ABSTRACT

The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.


Subject(s)
Intravital Microscopy/methods , RNA Polymerase II/metabolism , Single Molecule Imaging/methods , Transcription, Genetic , Genes, Reporter/genetics , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Microscopy, Fluorescence , Phosphorylation , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , Serine/metabolism , Spatio-Temporal Analysis , Time-Lapse Imaging
9.
Nat Struct Mol Biol ; 27(12): 1209-1210, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33110260

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

10.
Nat Struct Mol Biol ; 27(12): 1095-1104, 2020 12.
Article in English | MEDLINE | ID: mdl-32958947

ABSTRACT

Viruses use internal ribosome entry sites (IRES) to hijack host ribosomes and promote cap-independent translation. Although they are well-studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5' cap, and the other from the encephalomyocarditis virus IRES. When combined with a pair of complementary probes that bind the epitopes cotranslationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap-dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively, our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states.


Subject(s)
Encephalomyocarditis virus/genetics , Epithelial Cells/metabolism , Internal Ribosome Entry Sites , Protein Biosynthesis , RNA Caps/genetics , Base Pairing , Biosensing Techniques , Cell Line, Tumor , Encephalomyocarditis virus/metabolism , Epithelial Cells/virology , Epitopes/chemistry , Epitopes/genetics , Epitopes/metabolism , Host-Pathogen Interactions/genetics , Humans , Inverted Repeat Sequences , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Kinesins/genetics , Kinesins/metabolism , Molecular Probes/chemistry , Molecular Probes/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Open Reading Frames , RNA Caps/chemistry , RNA Caps/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Single Molecule Imaging/methods
11.
J Cell Biol ; 219(8)2020 08 03.
Article in English | MEDLINE | ID: mdl-32520986

ABSTRACT

Stress granules are dynamic assemblies of proteins and nontranslating RNAs that form when translation is inhibited in response to diverse stresses. Defects in ubiquitin-proteasome system factors including valosin-containing protein (VCP) and the proteasome impact the kinetics of stress granule induction and dissolution as well as being implicated in neuropathogenesis. However, the impacts of dysregulated proteostasis on mRNA regulation and stress granules are not well understood. Using single mRNA imaging, we discovered ribosomes stall on some mRNAs during arsenite stress, and the release of transcripts from stalled ribosomes for their partitioning into stress granules requires the activities of VCP, components of the ribosome-associated quality control (RQC) complex, and the proteasome. This is an unexpected contribution of the RQC system in releasing mRNAs from translation under stress, thus identifying a new type of stress-activated RQC (saRQC) distinct from canonical RQC pathways in mRNA substrates, cellular context, and mRNA fate.


Subject(s)
Cytoplasmic Granules/metabolism , Neoplasms/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Ribosomes/metabolism , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Arsenites/pharmacology , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/genetics , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Kinetics , Neoplasms/genetics , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Biosynthesis/drug effects , Protein Transport , Proteostasis , RNA, Messenger/genetics , Ribosomes/drug effects , Ribosomes/genetics , Sodium Compounds/pharmacology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolism
12.
PLoS Comput Biol ; 15(10): e1007425, 2019 10.
Article in English | MEDLINE | ID: mdl-31618265

ABSTRACT

Advances in fluorescence microscopy have introduced new assays to quantify live-cell translation dynamics at single-RNA resolution. We introduce a detailed, yet efficient sequence-based stochastic model that generates realistic synthetic data for several such assays, including Fluorescence Correlation Spectroscopy (FCS), ribosome Run-Off Assays (ROA) after Harringtonine application, and Fluorescence Recovery After Photobleaching (FRAP). We simulate these experiments under multiple imaging conditions and for thousands of human genes, and we evaluate through simulations which experiments are most likely to provide accurate estimates of elongation kinetics. Finding that FCS analyses are optimal for both short and long length genes, we integrate our model with experimental FCS data to capture the nascent protein statistics and temporal dynamics for three human genes: KDM5B, ß-actin, and H2B. Finally, we introduce a new open-source software package, RNA Sequence to NAscent Protein Simulator (rSNAPsim), to easily simulate the single-molecule translation dynamics of any gene sequence for any of these assays and for different assumptions regarding synonymous codon usage, tRNA level modifications, or ribosome pauses. rSNAPsim is implemented in Python and is available at: https://github.com/MunskyGroup/rSNAPsim.git.


Subject(s)
RNA, Messenger/metabolism , RNA/metabolism , Ribosomes/metabolism , Computational Biology/methods , Computer Simulation , Fluorescence Recovery After Photobleaching , Humans , Kinetics , Microscopy, Fluorescence , Protein Biosynthesis , Proteins/metabolism , RNA/physiology , Spectrometry, Fluorescence
13.
Nat Commun ; 10(1): 2947, 2019 07 03.
Article in English | MEDLINE | ID: mdl-31270320

ABSTRACT

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.


Subject(s)
Epitopes/metabolism , Molecular Probes/metabolism , Recombinant Fusion Proteins/metabolism , Single Molecule Imaging , Animals , Cell Line, Tumor , Embryo, Nonmammalian/metabolism , Green Fluorescent Proteins/metabolism , Neurons/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/isolation & purification , Single-Chain Antibodies/metabolism , Staining and Labeling , Zebrafish/embryology
14.
Mol Cell ; 75(1): 172-183.e9, 2019 07 11.
Article in English | MEDLINE | ID: mdl-31178355

ABSTRACT

Ribosomal frameshifting during the translation of RNA is implicated in human disease and viral infection. While previous work has uncovered many details about single RNA frameshifting kinetics in vitro, little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames. Applying this technology to RNA encoding the HIV-1 frameshift sequence reveals a small subset (∼8%) of the translating pool robustly frameshift. Frameshifting RNA are translated at similar rates as non-frameshifting RNA (∼3 aa/s) and can continuously frameshift for more than four rounds of translation. Fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide insight into retroviral frameshifting and could lead to alternative strategies to perturb the process in living cells.


Subject(s)
Frameshifting, Ribosomal , HIV-1/genetics , Open Reading Frames , Osteoblasts/metabolism , RNA, Viral/genetics , Single Molecule Imaging/methods , Base Pairing , Cell Line, Tumor , HIV-1/metabolism , Humans , Models, Genetic , Nucleic Acid Conformation , Oligonucleotide Probes/chemical synthesis , Oligonucleotide Probes/genetics , Oligonucleotide Probes/metabolism , Oligopeptides/genetics , Oligopeptides/metabolism , Osteoblasts/virology , RNA, Viral/chemistry , RNA, Viral/metabolism , Staining and Labeling/methods
15.
Nat Cell Biol ; 21(2): 162-168, 2019 02.
Article in English | MEDLINE | ID: mdl-30664789

ABSTRACT

Ribonucleoprotein (RNP) granules are non-membrane-bound organelles that have critical roles in the stress response1,2, maternal messenger RNA storage3, synaptic plasticity4, tumour progression5,6 and neurodegeneration7-9. However, the dynamics of their mRNA components within and near the granule surface remain poorly characterized, particularly in the context and timing of mRNAs exiting translation. Herein, we used multicolour single-molecule tracking to quantify the precise timing and kinetics of single mRNAs as they exit translation and enter RNP granules during stress. We observed single mRNAs interacting with stress granules and P-bodies, with mRNAs moving bidirectionally between them. Although translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable, and sometimes rigid, associations with RNP granules with stability increasing with both mRNA length and granule size. Live and fixed cell imaging demonstrated that mRNAs can extend beyond the protein surface of a stress granule, which may facilitate interactions between RNP granules. Thus, the recruitment of mRNPs to RNP granules involves dynamic, stable and extended interactions affected by translation status, mRNA length and granule size that collectively regulate RNP granule dynamics.


Subject(s)
Cell Tracking/methods , Cytoplasmic Granules/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Cell Line, Tumor , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Kinetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Biological , Protein Binding , RNA, Messenger/genetics , Ribonucleoproteins/genetics , Stress, Physiological , Time Factors , Red Fluorescent Protein
16.
Article in English | MEDLINE | ID: mdl-30385605

ABSTRACT

One of the last hurdles to quantifying the full central dogma of molecular biology in living cells with single-molecule resolution has been the imaging of single messenger RNA (mRNA) translation. Here we describe how recent advances in protein tagging and imaging technologies are being used to precisely visualize and quantify the synthesis of nascent polypeptide chains from single mRNA in living cells. We focus on recent applications of repeat-epitope tags and describe how they enable quantification of single mRNA ribosomal densities, translation initiation and elongation rates, and translation site mobility and higher-order structure. Together with complementary live-cell assays to monitor translation using fast-maturing fluorophores and mRNA-binding protein knockoff, single-molecule studies are beginning to uncover striking and unexpected heterogeneity in gene expression at the level of translation.


Subject(s)
Molecular Imaging/methods , Protein Biosynthesis/physiology , RNA, Messenger/metabolism , Epitopes , Gene Expression Regulation , Humans , Peptide Chain Elongation, Translational
17.
Nat Commun ; 8(1): 2151, 2017 12 15.
Article in English | MEDLINE | ID: mdl-29247176

ABSTRACT

Cytoplasmic dynein is an enormous minus end-directed microtubule motor. Rather than existing as bare tracks, microtubules are bound by numerous microtubule-associated proteins (MAPs) that have the capacity to affect various cellular functions, including motor-mediated transport. One such MAP is She1, a dynein effector that polarizes dynein-mediated spindle movements in budding yeast. Here, we characterize the molecular basis by which She1 affects dynein, providing the first such insight into which a MAP can modulate motor motility. We find that She1 affects the ATPase rate, microtubule-binding affinity, and stepping behavior of dynein, and that microtubule binding by She1 is required for its effects on dynein motility. Moreover, we find that She1 directly contacts the microtubule-binding domain of dynein, and that their interaction is sensitive to the nucleotide-bound state of the motor. Our data support a model in which simultaneous interactions between the microtubule and dynein enables She1 to directly affect dynein motility.


Subject(s)
Dyneins/metabolism , Microtubules/metabolism , Myosin Heavy Chains/metabolism , Myosin Type V/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Sequence , Dyneins/chemistry , Dyneins/genetics , Microscopy, Fluorescence , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Models, Biological , Models, Molecular , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/genetics , Molecular Motor Proteins/metabolism , Myosin Heavy Chains/chemistry , Myosin Heavy Chains/genetics , Myosin Type V/chemistry , Myosin Type V/genetics , Protein Binding , Protein Domains , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
18.
Nat Commun ; 8(1): 313, 2017 08 22.
Article in English | MEDLINE | ID: mdl-28827596

ABSTRACT

Live-cell microscopy has highlighted that transcription factors bind transiently to chromatin but it is not clear if the duration of these binding interactions can be modulated in response to an activation stimulus, and if such modulation can be controlled by post-translational modifications of the transcription factor. We address this question for the tumor suppressor p53 by combining live-cell single-molecule microscopy and single cell in situ measurements of transcription and we show that p53-binding kinetics are modulated following genotoxic stress. The modulation of p53 residence times on chromatin requires C-terminal acetylation-a classical mark for transcriptionally active p53-and correlates with the induction of transcription of target genes such as CDKN1a. We propose a model in which the modification state of the transcription factor determines the coupling between transcription factor abundance and transcriptional activity by tuning the transcription factor residence time on target sites.Both transcription binding kinetics and post-translational modifications of transcription factors are thought to play a role in the modulation of transcription. Here the authors use single-molecule tracking to directly demonstrate that p53 acetylation modulates promoter residence time and transcriptional activity.


Subject(s)
Cell Tracking/methods , Promoter Regions, Genetic/genetics , Transcription, Genetic , Tumor Suppressor Protein p53/metabolism , Acetylation , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Humans , MCF-7 Cells , Microscopy, Confocal , Protein Binding , Time Factors , Transcription Factors/metabolism , Tumor Suppressor Protein p53/genetics
19.
Nat Commun ; 8: 15896, 2017 06 21.
Article in English | MEDLINE | ID: mdl-28635963

ABSTRACT

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other's dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level.


Subject(s)
Molecular Imaging/methods , Receptors, Steroid/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Chromatin/metabolism , Corticosterone/pharmacology , DNA Helicases/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/genetics , Mice , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Protein Interaction Mapping , Rats , Receptors, Glucocorticoid/analysis , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Receptors, Steroid/analysis , Receptors, Steroid/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Single-Cell Analysis/methods , Time Factors , Transcription Factor AP-1/metabolism , Transcription Factors/metabolism
20.
Nucleic Acids Res ; 44(21): e160, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27566148

ABSTRACT

In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12-0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.


Subject(s)
Chromatin/metabolism , DNA-Binding Proteins/analysis , DNA-Binding Proteins/metabolism , Molecular Imaging/methods , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/analysis , Transcription Factors/metabolism , DNA-Binding Proteins/genetics , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Histones/genetics , Histones/metabolism , Metallothionein/genetics , Metallothionein/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Time Factors , Transcription Factors/genetics
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