scholarly journals DNA sequence encodes the position of DNA supercoils

2017 ◽  
Author(s):  
Sung Hyun Kim ◽  
Mahipal Ganji ◽  
Jaco van der Torre ◽  
Elio Abbondanzieri ◽  
Cees Dekker
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Dna Sequences ◽  
Structural Information ◽  
Three Dimensional ◽  
Decisive Role ◽  
Dimensional Structure ◽  
Transcription Start Sites ◽  
Cellular Processes

AbstractThe three-dimensional structure of DNA is increasingly understood to play a decisive role in gene regulation and other vital cellular processes, which has triggered an explosive growth of research on the spatial architecture of the genome. Many studies focus on the role of various DNA-packaging proteins, crowding, and confinement in organizing chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we use a fluorescence-based single-molecule technique to visualize plectonemes, the extended intertwined DNA loops that form upon twisting DNA. Remarkably, we find that the underlying DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific positions. We explore a variety of DNA sequences to determine what features influence pinning, and we develop a physical model that predicts the level of plectoneme pinning in excellent agreement with the data. The intrinsic curvature measured over a range of ~70 base pairs is found to be the key property governing the supercoiled structure of DNA. Our model predicts that plectonemes are likely to localize directly upstream of prokaryotic transcription start sites, and this prediction is experimentally verifiedin vitro.Our results reveal a hidden code in DNA that helps to spatially organize the genome.

scholarly journals DNA sequence encodes the position of DNA supercoils

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sung Hyun Kim ◽  
Mahipal Ganji ◽  
Eugene Kim ◽  
Jaco van der Torre ◽  
Elio Abbondanzieri ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Structural Information ◽  
Three Dimensional ◽  
Decisive Role ◽  
Chromosomal Dna ◽  
Dna Structures ◽  
Cellular Processes ◽  
Specific Sequences ◽  
Good Agreement

The three-dimensional organization of DNA is increasingly understood to play a decisive role in vital cellular processes. Many studies focus on the role of DNA-packaging proteins, crowding, and confinement in arranging chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we visualize plectonemes (extended intertwined DNA structures formed upon supercoiling) on individual DNA molecules. Remarkably, our experiments show that the DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific sequences. We develop a physical model that predicts that sequence-dependent intrinsic curvature is the key determinant of pinning strength and demonstrate this simple model provides very good agreement with the data. Analysis of several prokaryotic genomes indicates that plectonemes localize directly upstream of promoters, which we experimentally confirm for selected promotor sequences. Our findings reveal a hidden code in the genome that helps to spatially organize the chromosomal DNA.

Unfolding single RNA molecules: bridging the gap between equilibrium and non-equilibrium statistical thermodynamics

2005 ◽  
Vol 38 (4) ◽  
pp. 291-301 ◽  
Author(s):  
Carlos Bustamante
Keyword(s):  
Single Molecule ◽  
Molecular Motors ◽  
Biological Systems ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Fluctuation Theorems ◽  
Rna Molecules ◽  
The Times ◽  
Non Equilibrium

During the last 15 years, scientists have developed methods that permit the direct mechanical manipulation of individual molecules. Using this approach, they have begun to investigate the effect of force and torque in chemical and biochemical reactions. These studies span from the study of the mechanical properties of macromolecules, to the characterization of molecular motors, to the mechanical unfolding of individual proteins and RNA. Here I present a review of some of our most recent results using mechanical force to unfold individual molecules of RNA. These studies make it possible to follow in real time the trajectory of each molecule as it unfolds and characterize the various intermediates of the reaction. Moreover, if the process takes place reversibly it is possible to extract both kinetic and thermodynamic information from these experiments at the same time that we characterize the forces that maintain the three-dimensional structure of the molecule in solution. These studies bring us closer to the biological unfolding processes in the cell as they simulate in vitro, the mechanical unfolding of RNAs carried out in the cell by helicases. If the unfolding process occurs irreversibly, I show here that single-molecule experiments can still provide equilibrium, thermodynamic information from non-equilibrium data by using recently discovered fluctuation theorems. Such theorems represent a bridge between equilibrium and non-equilibrium statistical mechanics. In fact, first derived in 1997, the first experimental demonstration of the validity of fluctuation theorems was obtained by unfolding mechanically a single molecule of RNA. It is perhaps a sign of the times that important physical results are these days used to extract information about biological systems and that biological systems are being used to test and confirm fundamental new laws in physics.

scholarly journals Microtubule architecture in vitro and in cells revealed by cryo-electron tomography

2018 ◽  
Vol 74 (6) ◽  
pp. 572-584 ◽  
Author(s):  
Joseph Atherton ◽  
Melissa Stouffer ◽  
Fiona Francis ◽  
Carolyn A. Moores
Keyword(s):  
Molecular Motors ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
In Cells

The microtubule cytoskeleton is involved in many vital cellular processes. Microtubules act as tracks for molecular motors, and their polymerization and depolymerization can be harnessed to generate force. The structures of microtubules provide key information about the mechanisms by which their cellular roles are accomplished and the physiological context in which these roles are performed. Cryo-electron microscopy allows the visualization of in vitro-polymerized microtubules and has provided important insights into their overall morphology and the influence of a range of factors on their structure and dynamics. Cryo-electron tomography can be used to determine the unique three-dimensional structure of individual microtubules and their ends. Here, a previous cryo-electron tomography study of in vitro-polymerized GMPCPP-stabilized microtubules is revisited, the findings are compared with new tomograms of dynamic in vitro and cellular microtubules, and the information that can be extracted from such data is highlighted. The analysis shows the surprising structural heterogeneity of in vitro-polymerized microtubules. Lattice defects can be observed both in vitro and in cells. The shared ultrastructural properties in these different populations emphasize the relevance of three-dimensional structures of in vitro microtubules for understanding microtubule cellular functions.

scholarly journals The lighthouse at the end of the chromosome*

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1427 ◽  
Author(s):  
Yahya Benslimane ◽  
Lea Harrington
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Spatial Organization ◽  
Three Dimensional ◽  
Time Lapse ◽  
Living Cells ◽  
Telomere Elongation ◽  
Cellular Processes ◽  
Time Lapse Microscopy

Fluorescence microscopy can be used to assess the dynamic localization and intensity of single entities in vitro or in living cells. It has been applied with aplomb to many different cellular processes and has significantly enlightened our understanding of the heterogeneity and complexity of biological systems. Recently, high-resolution fluorescence microscopy has been brought to bear on telomeres, leading to new insights into telomere spatial organization and accessibility, and into the mechanistic nuances of telomere elongation. We provide a snapshot of some of these recent advances with a focus on mammalian systems, and show how three-dimensional, time-lapse microscopy and single-molecule fluorescence shine a new light on the end of the chromosome.

Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 282-283
Author(s):  
José L. Carrascosa ◽  
José M. Valpuesta ◽  
Hisao Fujisawa
Keyword(s):  
Dna Sequences ◽  
Expression Vector ◽  
Three Dimensional ◽  
Deae Cellulose ◽  
Dna Packaging ◽  
Dimensional Structure ◽  
Two Dimensional ◽  
Freezing And Thawing ◽  
Three Dimensional Structure ◽  
Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

scholarly journals Nanofiber-Mâché Hollow Ball Mimicking the Three-Dimensional Structure of a Cyst

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2273
Author(s):  
Wan-Ying Huang ◽  
Norichika Hashimoto ◽  
Ryuhei Kitai ◽  
Shin-ichiro Suye ◽  
Satoshi Fujita
Keyword(s):  
Three Dimensional ◽  
Hollow Structure ◽  
Dimensional Structure ◽  
The Novel ◽  
Fibrous Scaffold ◽  
Hydrogel Beads ◽  
Inner Surface ◽  
Intracranial Epidermoid ◽  
Hollow Ball

The occasional malignant transformation of intracranial epidermoid cysts into squamous cell carcinomas remains poorly understood; the development of an in vitro cyst model is urgently needed. For this purpose, we designed a hollow nanofiber sphere, the “nanofiber-mâché ball.” This hollow structure was fabricated by electrospinning nanofiber onto alginate hydrogel beads followed by dissolving the beads. A ball with approximately 230 mm3 inner volume provided a fibrous geometry mimicking the topography of the extracellular matrix. Two ducts located on opposite sides provided a route to exchange nutrients and waste. This resulted in a concentration gradient that induced oriented migration, in which seeded cells adhered randomly to the inner surface, formed a highly oriented structure, and then secreted a dense web of collagen fibrils. Circumferentially aligned fibers on the internal interface between the duct and hollow ball inhibited cells from migrating out of the interior, similar to a fish bottle trap. This structure helped to form an adepithelial layer on the inner surface. The novel nanofiber-mâché technique, using a millimeter-sized hollow fibrous scaffold, is excellently suited to investigating cyst physiology.

Three-Dimensional High Resolution Scaffold Fiber Architecture and Morphology in Tissue Engineered Heart Valve Tissue

2010 ◽  
Author(s):  
Chad E. Eckert ◽  
Brandon T. Mikulis ◽  
Dane Gerneke ◽  
Danielle Gottlieb ◽  
Bruce Smaill ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
High Resolution ◽  
Heart Valve ◽  
Structural Information ◽  
Three Dimensional ◽  
Tissue Scaffold ◽  
Valve Tissue ◽  
Sampling Protocols ◽  
Heart Valve Tissue

Engineered heart valve tissue (EHVT) has received much attention as a potential pediatric valve replacement therapy, offering prospective long-term functional improvements over current options. A significant gap in the literature exists, however, regarding estimating tissue mechanical properties from tissue-scaffold composites. Detailed three-dimensional structural information prior to implantation (in vitro) and after implantation in (in vivo) is needed for improved modeling of tissue properties. As such, a novel high-resolution imaging technique will be employed to obtain three-dimensional microstructural information. Analysis techniques will be used to fully quantify constituents of interest including scaffold, collagen, and cellular information and to develop appropriate two-dimensional sectioning sampling protocols. It is the intent of this work to guide modeling efforts to better elucidate EHVT tissue-specific mechanical properties.

scholarly journals Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

2008 ◽  
Vol 190 (6) ◽  
pp. 2056-2064 ◽  
Author(s):  
Jonathan E. Ulmer ◽  
Yap Boum ◽  
Christopher D. Thouvenel ◽  
Hannu Myllykallio ◽  
Carol Hopkins Sibley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Thymidylate Synthase ◽  
Three Dimensional ◽  
Pyrimidine Ring ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Catalytic Residue ◽  
Amino Acid Residues ◽  
Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

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