CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPgS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum-dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.

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Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading from parS

10.1101/2021.02.11.430778 ◽

2021 ◽

Author(s):

Francisco de Asis Balaguer ◽

Clara Aicart-Ramos ◽

Gemma LM Fisher ◽

Sara de Bragança ◽

Cesar L. Pastrana ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Dna Sequences ◽

Specific Interaction ◽

Magnetic Tweezers ◽

One Dimensional ◽

Sliding Clamp ◽

Absolute Requirement ◽

Bacterial Chromosomes

SUMMARYFaithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined dual optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced 4-fold by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific regions of DNA. This requires the presence of a parS loading site and is prevented by roadblocks on DNA, consistent with one dimensional diffusion by a sliding clamp. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations. We propose a model in which ParB-CTP-Mg2+ complexes move along DNA following loading at parS sites and protein:protein interactions result in the localised condensation of DNA within ParB networks.

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Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

10.1101/854968 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alberto Marin-Gonzalez ◽

Cesar L. Pastrana ◽

Rebeca Bocanegra ◽

Alejandro Martín-González ◽

J.G. Vilhena ◽

...

Keyword(s):

Mechanical Properties ◽

Optical Tweezers ◽

Single Molecule ◽

Dna Sequences ◽

Mechanical Response ◽

Magnetic Tweezers ◽

Crystallographic Structure ◽

Chain Model ◽

Microscopy Imaging ◽

Length Scales

ABSTRACTA-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, their response to forces remains unknown and the variability of their flexibility reported for different length scales has precluded a comprehensive description of the mechanical properties of these molecules. Here, we rationalize the mechanical properties of A-tracts across multiple length scales using a combination of single-molecule experiments and theoretical polymer models applied to DNA sequences present in the C. elegans genome. Atomic Force Microscopy imaging shows that phased A-tracts induce long-range (∼200 nm) bending. Moreover, the enhanced bending originates from an intrinsically bent structure rather than as a consequence of larger flexibility. In support of this, our data were well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments confirm that the observed bent is intrinsic to the sequence and does not rely on particular ionic conditions. Using optical tweezers, we assess the local rigidity of A-tracts at high forces and unravel an unusually stiff character of these sequences, as quantified by their large stretch modulus. Our work rationalizes the complex multiscale flexibility of A-tracts, shedding light on the cryptic character of these sequences.

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Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

Nucleic Acids Research ◽

10.1093/nar/gkaa225 ◽

2020 ◽

Vol 48 (9) ◽

pp. 5024-5036

Author(s):

Alberto Marin-Gonzalez ◽

Cesar L Pastrana ◽

Rebeca Bocanegra ◽

Alejandro Martín-González ◽

J G Vilhena ◽

...

Keyword(s):

Mechanical Properties ◽

Optical Tweezers ◽

Single Molecule ◽

Dna Sequences ◽

Mechanical Response ◽

Magnetic Tweezers ◽

Crystallographic Structure ◽

Chain Model ◽

Microscopy Imaging ◽

Wide Range

Abstract A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

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Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

International Journal of Molecular Sciences ◽

10.3390/ijms22052398 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2398

Author(s):

Wooyoung Kang ◽

Seungha Hwang ◽

Jin Young Kang ◽

Changwon Kang ◽

Sungchul Hohng

Keyword(s):

Single Molecule ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Facilitated Diffusion ◽

One Dimensional ◽

Bacterial Transcription ◽

Fluorescence Measurements ◽

Dimensional Diffusion ◽

Single Molecule Studies ◽

Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

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Bio-Molecular Applications of Recent Developments in Optical Tweezers

Biomolecules ◽

10.3390/biom9010023 ◽

2019 ◽

Vol 9 (1) ◽

pp. 23 ◽

Cited By ~ 17

Author(s):

Dhawal Choudhary ◽

Alessandro Mossa ◽

Milind Jadhav ◽

Ciro Cecconi

Keyword(s):

Photonic Crystal ◽

Optical Tweezers ◽

Single Molecule ◽

Continuous Wave ◽

Imaging Techniques ◽

Resolving Power ◽

Optical Traps ◽

Laser Source ◽

One Dimensional

In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

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DESIGNED SELF-ORGANIZATION FOR MOLECULAR OPTOELECTRONIC SENSORS

International Journal of High Speed Electronics and Systems ◽

10.1142/s0129156407004527 ◽

2007 ◽

Vol 17 (02) ◽

pp. 311-326 ◽

Cited By ~ 3

Author(s):

Michael Norton

Keyword(s):

Single Molecule ◽

Dna Sequences ◽

Terahertz Spectroscopy ◽

Attachment Site ◽

Surface Immobilization ◽

Surface Attachment ◽

One Dimensional ◽

Synthetic Dna ◽

Attachment Sites ◽

Sensitivity And Selectivity

The convergence of terahertz spectroscopy and single molecule experimentation offers significant promise of enhancement in sensitivity and selectivity in molecular recognition, identification and quantitation germane to military and security applications. This paper provides a brief overview of the constraints set by single molecule recognition systems and reports the results of experiments which address fundamental barriers to the integration of large, patterned bio-compatible molecular opto-electronic systems with silicon based microelectronic systems. Central to this thrust is an approach involving sequential epitaxy on surface bound single stranded DNA one-dimensional substrates. The challenge of producing highly structured macromolecular substrates, which are necessary in order to implement molecular nanolithography, has been addressed experimentally by combining “designer” synthetic DNA with biosynthetically derived plasmid components. By design, these one dimensional templates are composed of domains which contain sites which are recognized, and therefore addressable by either complementary DNA sequences and/or selected enzymes. Such design is necessary in order to access the nominal 2 nm linewidth potential resolution of nanolithography on these one-dimensional substrates. The recognition and binding properties of DNA ensure that the lithographic process is intrinsically self-organizing, and therefore self-aligning, a necessity for assembly processes at the requisite resolution. Another requirement of this molecular epitaxy approach is that the substrate must be immobilized. The challenge of robust surface immobilization is being addressed via the production of the equivalent of molecular tube sockets. In this application, multi-valent core-shell fluorescent quantum dots provide a mechanism to prepare surface attachment sites with a pre-determined 1:1 attachment site : substrate (DNA) molecule ratio.

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Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence

Nucleic Acids Research ◽

10.1093/nar/gkaa875 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11602-11614

Author(s):

Youbin Mo ◽

Nicholas Keller ◽

Damian delToro ◽

Neeti Ananthaswamy ◽

Stephen C Harvey ◽

...

Keyword(s):

Dna Sequence ◽

Motor Function ◽

Optical Tweezers ◽

Single Molecule ◽

Dna Sequences ◽

Viral Genome ◽

Bacteriophage T4 ◽

Repeated Measurements ◽

Genome Packaging ◽

Sequence Dependence

Abstract Many viruses employ ATP-powered motors during assembly to translocate DNA into procapsid shells. Previous reports raise the question if motor function is modulated by substrate DNA sequence: (i) the phage T4 motor exhibits large translocation rate fluctuations and pauses and slips; (ii) evidence suggests that the phage phi29 motor contacts DNA bases during translocation; and (iii) one theoretical model, the ‘B-A scrunchworm’, predicts that ‘A-philic’ sequences that transition more easily to A-form would alter motor function. Here, we use single-molecule optical tweezers measurements to compare translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor. We observed no significant differences in motor velocities, even with A-philic sequences predicted to show higher translocation rate at high applied force. We also observed no significant changes in motor pausing and only modest changes in slipping. To more generally test for sequence dependence, we conducted correlation analyses across pairs of packaging events. No significant correlations in packaging rate, pausing or slipping versus sequence position were detected across repeated measurements with several different DNA sequences. These studies suggest that viral genome packaging is insensitive to DNA sequence and fluctuations in packaging motor velocity, pausing and slipping are primarily stochastic temporal events.

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Bioconjugation Strategies for Connecting Proteins to DNA-Linkers for Single-Molecule Force-Based Experiments

Nanomaterials ◽

10.3390/nano11092424 ◽

2021 ◽

Vol 11 (9) ◽

pp. 2424

Author(s):

Lyan M. van der Sleen ◽

Katarzyna M. Tych

Keyword(s):

Mechanical Properties ◽

Atomic Force Microscopy ◽

Optical Tweezers ◽

Single Molecule ◽

Short Range ◽

Force Spectroscopy ◽

Magnetic Tweezers ◽

Single Molecule Force Spectroscopy ◽

Force Microscopy ◽

Atomic Force

The mechanical properties of proteins can be studied with single molecule force spectroscopy (SMFS) using optical tweezers, atomic force microscopy and magnetic tweezers. It is common to utilize a flexible linker between the protein and trapped probe to exclude short-range interactions in SMFS experiments. One of the most prevalent linkers is DNA due to its well-defined properties, although attachment strategies between the DNA linker and protein or probe may vary. We will therefore provide a general overview of the currently existing non-covalent and covalent bioconjugation strategies to site-specifically conjugate DNA-linkers to the protein of interest. In the search for a standardized conjugation strategy, considerations include their mechanical properties in the context of SMFS, feasibility of site-directed labeling, labeling efficiency, and costs.

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Solid-State Nanopores: From Fabrication to Application

Microscopy Today ◽

10.1017/s1551929512000703 ◽

2012 ◽

Vol 20 (5) ◽

pp. 24-29 ◽

Cited By ~ 2

Author(s):

Adam R. Hall

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

High Speed ◽

Super Resolution ◽

Magnetic Tweezers ◽

Fluorescent Imaging ◽

Force Microscopy ◽

Atomic Force ◽

Labeling Requirements ◽

Molecular Manipulation

There are relatively few technologies for measurement at the single-molecule scale. Fluorescent imaging, for example, can be used to directly visualize molecules and their interactions, but diffraction limitations and labeling requirements may push the system from its native state. Although recent advances in super-resolution imaging have been able to break this resolution barrier, important challenges remain. Atomic force microscopy (AFM) is capable of imaging molecules at high resolution and at high speed. However, AFM imaging is a surface technique, requiring sample preparation and some immobilization. Other technologies such as optical tweezers and magnetic tweezers are capable of molecular manipulation and spectroscopy to great effect but require a significant apparatus and have limited inherent analytical capabilities.

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Broad distributions of transition-path times are fingerprints of multidimensionality of the underlying free energy landscapes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008307117 ◽

2020 ◽

Vol 117 (44) ◽

pp. 27116-27123 ◽

Cited By ~ 1

Author(s):

Rohit Satija ◽

Alexander M. Berezhkovskii ◽

Dmitrii E. Makarov

Keyword(s):

Free Energy ◽

Single Molecule ◽

Coefficient Of Variation ◽

Biological Membrane ◽

Energy Landscape ◽

Transition Path ◽

Activation Barriers ◽

One Dimensional ◽

Dimensional Diffusion ◽

Transition Paths

Recent single-molecule experiments have observed transition paths, i.e., brief events where molecules (particularly biomolecules) are caught in the act of surmounting activation barriers. Such measurements offer unprecedented mechanistic insights into the dynamics of biomolecular folding and binding, molecular machines, and biological membrane channels. A key challenge to these studies is to infer the complex details of the multidimensional energy landscape traversed by the transition paths from inherently low-dimensional experimental signals. A common minimalist model attempting to do so is that of one-dimensional diffusion along a reaction coordinate, yet its validity has been called into question. Here, we show that the distribution of the transition path time, which is a common experimental observable, can be used to differentiate between the dynamics described by models of one-dimensional diffusion from the dynamics in which multidimensionality is essential. Specifically, we prove that the coefficient of variation obtained from this distribution cannot possibly exceed 1 for any one-dimensional diffusive model, no matter how rugged its underlying free energy landscape is: In other words, this distribution cannot be broader than the single-exponential one. Thus, a coefficient of variation exceeding 1 is a fingerprint of multidimensional dynamics. Analysis of transition paths in atomistic simulations of proteins shows that this coefficient often exceeds 1, signifying essential multidimensionality of those systems.

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