Modern biophysical approaches probe transcription-factor-induced DNA bending and looping

2013 ◽  
Vol 41 (1) ◽  
pp. 368-373 ◽  
Author(s):  
Andreas Gietl ◽  
Dina Grohmann
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Conformational Changes ◽  
Double Helix ◽  
Dna Bending ◽  
Living Organism ◽  
Single Molecule Level ◽  
Specific Manner ◽  
Biophysical Techniques ◽  
Promoter Dna

The genetic information of every living organism is stored in its genomic DNA that is perceived as a chemically stable and robust macromolecule. But at the same time, to fulfil its functions properly, it also needs to be highly dynamic and flexible. This includes partial melting of the double helix or compaction and bending of the DNA often brought about by protein factors that are able to interact with DNA stretches in a specific and non-specific manner. The conformational changes in the DNA need to be understood in order to describe biological systems in detail. As these events play out on the nanometre scale, new biophysical approaches have been employed to monitor conformational changes in this regime at the single-molecule level. Focusing on transcription factor action on promoter DNA, we discuss how current biophysical techniques are able to quantitatively describe this molecular process.

scholarly journals Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model

2021 ◽  
Author(s):  
David A Garcia ◽  
Gregory Fettweis ◽  
Diego M Presman ◽  
Ville Paakinaho ◽  
Christopher Jarzynski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Power Law ◽  
Dwell Time ◽  
Specific Binding ◽  
Power Law Distribution ◽  
Single Molecule Level ◽  
Binding Behavior ◽  
Chromatin Template ◽  
Nuclear Domains

Abstract Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

scholarly journals Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Abhishek Mazumder ◽  
Richard H Ebright ◽  
Achillefs Kapanidis
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Extensive Study ◽  
Active Centre ◽  
Transient Nature ◽  
Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

scholarly journals Microsecond and millisecond dynamics in the photosynthetic protein LHCSR1 observed by single-molecule correlation spectroscopy

2019 ◽  
Vol 116 (23) ◽  
pp. 11247-11252 ◽  
Author(s):  
Toru Kondo ◽  
Jesse B. Gordon ◽  
Alberta Pinnola ◽  
Luca Dall’Osto ◽  
Roberto Bassi ◽  
...  
Keyword(s):  
Correlation Function ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Light Harvesting ◽  
Building Blocks ◽  
Complex Stress ◽  
Correlation Spectroscopy ◽  
Light Harvesting Complex ◽  
Single Molecule Level

Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function.

scholarly journals Transcription Factor Target Search Strategy at the Single Molecule Level in Eukaryotic Cells

2012 ◽  
Vol 102 (3) ◽  
pp. 288a
Author(s):  
Lydia Boudarene ◽  
Vincent Récamier ◽  
Davide Normanno ◽  
Ignacio Izzedine ◽  
Ibrahim Cissé ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Search Strategy ◽  
Eukaryotic Cells ◽  
Target Search ◽  
Single Molecule Level ◽  
Transcription Factor Target

scholarly journals Membrane Active Peptides and Their Biophysical Characterization

2018 ◽  
Author(s):  
Fatma Gizem Avci ◽  
Berna Sariyar Akbulut ◽  
Elif Ozkirimli
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Membrane Integrity ◽  
Drug Market ◽  
Cell Penetrating Peptides ◽  
Single Molecule Level ◽  
Active Peptides ◽  
Biophysical Techniques ◽  
Membrane Active Peptides ◽  
Dynamics Simulations

In the last 20 years, an increasing number of studies have been reported on membrane active peptides, which exert their biological activity by interacting with the cell membrane either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. These peptides are attractive alternatives to currently used pharmaceuticals. Antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery currently in the drug pipeline suggest that these membrane active peptides will soon constitute a significant percentage of the drug market. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). AMPs are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus and fungi. CPPs are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity to some extent. Biophysical techniques to understand how they interact with the membrane have shed light on the peptide–membrane interaction at various levels of detail. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Advances in live imaging techniques have allowed examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provided clues on the peptide-lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.

scholarly journals Optical imaging of single protein size, charge, mobility, binding and conformational change

2018 ◽  
Author(s):  
Guanzhong Ma ◽  
Hao Zhu ◽  
Zijian Wan ◽  
Yunze Yang ◽  
Shaopeng Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Near Field ◽  
Protein Analysis ◽  
Imaging Method ◽  
Charge Mobility ◽  
Single Molecule Level ◽  
Protein Size ◽  
Flexible Polymer ◽  
Protein Oscillation

AbstractProtein analysis has relied on electrophoresis, mass spectroscopy and immunoassay, which separate, detect and identify proteins based on the size, charge, mobility and binding to antibodies. However, measuring these quantities at the single molecule level has not been possible. We tether a protein to a surface with a flexible polymer, drive the protein into mechanical oscillation with an alternating electric field, and image the protein oscillation with a near field imaging method, from which we determine the size, charge, mobility of the protein. We also measure binding of antibodies to single proteins and ligand binding-induced conformational changes in single proteins. This work provides new capabilities for protein analysis and disease biomarker detection at the single molecule level.

scholarly journals The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation

2020 ◽  
Vol 48 (5) ◽  
pp. 2604-2620 ◽  
Author(s):  
Urmimala Basu ◽  
Seung-Won Lee ◽  
Aishwarya Deshpande ◽  
Jiayu Shen ◽  
Byeong-Kwon Sohn ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Initiation Factor ◽  
Mitochondrial Rna ◽  
Elongation Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Template Strand ◽  
Mitochondrial Transcription Factor ◽  
Promoter Dna

Abstract Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA–DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA–DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

scholarly journals Membrane Active Peptides and Their Biophysical Characterization

Biomolecules ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 77 ◽  
Author(s):  
Fatma Gizem Avci ◽  
Berna Sariyar Akbulut ◽  
Elif Ozkirimli
Keyword(s):  
Antimicrobial Peptides ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Membrane Integrity ◽  
Cell Penetrating Peptides ◽  
Single Molecule Level ◽  
Active Peptides ◽  
Cell Penetrating ◽  
Biophysical Techniques ◽  
Membrane Active Peptides

In the last 20 years, an increasing number of studies have been reported on membrane active peptides. These peptides exert their biological activity by interacting with the cell membrane, either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. Membrane active peptides are attractive alternatives to currently used pharmaceuticals and the number of antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery in the drug pipeline is increasing. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). Antimicrobial peptides are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus, and fungi. Cell penetrating peptides are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity. Biophysical techniques shed light on peptide–membrane interactions at higher resolution due to the advances in optics, image processing, and computational resources. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Live imaging techniques allow examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provide clues on the peptide–lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.

scholarly journals Quantifying transcription factor binding dynamics at the single-molecule level in live cells

Methods ◽  
2017 ◽  
Vol 123 ◽  
pp. 76-88 ◽  
Author(s):  
Diego M. Presman ◽  
David A. Ball ◽  
Ville Paakinaho ◽  
Jonathan B. Grimm ◽  
Luke D. Lavis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Transcription Factor Binding ◽  
Live Cells ◽  
Single Molecule Level ◽  
Factor Binding

scholarly journals Power-law behaviour of transcription factor dynamics at the single-molecule level implies a continuum affinity model

2019 ◽  
Author(s):  
David A. Garcia ◽  
Gregory Fettweis ◽  
Diego M. Presman ◽  
Ville Paakinaho ◽  
Christopher Jarzynski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Power Law ◽  
Specific Binding ◽  
Power Law Distribution ◽  
Single Molecule Level ◽  
Complex Interactions ◽  
Chromatin Template ◽  
Nuclear Domains

ABSTRACTSingle-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the search and binding behaviour of these proteins in the nuclear environment. Dwell time distributions for most TFs have been described by SMT to follow bi-exponential behaviour. This is consistent with the existence of two discrete populations bound to chromatin in vivo, one non-specifically bound to chromatin (i.e. searching mode) and another specifically bound to target sites, as originally defined by decades of biochemical studies. However, alternative models have started to emerge, from multiple exponential components to power-law distributions. Here, we present an analytical pipeline with an unbiased model selection approach based on different statistical metrics to determine the model that best explains SMT data. We found that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution, blurring the temporal line between non-specific and specific binding, and suggesting that productive binding may involve longer binding events than previously thought. We propose a continuum of affinities model to explain the experimental data, consistent with the movement of TFs through complex interactions with multiple nuclear domains as well as binding and searching on the chromatin template.

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