scholarly journals Designed architectural proteins that tune DNA looping in bacteria

2021 ◽  
Author(s):  
David H. Tse ◽  
Nicole A. Becker ◽  
Robert T. Young ◽  
Wilma K. Olson ◽  
Justin P. Peters ◽  
...  
Keyword(s):  
Protein Binding ◽  
Double Helix ◽  
Dna Bending ◽  
Lac Repressor ◽  
Dna Looping ◽  
Transcription Activator ◽  
Dna Loops ◽  
Bent Dna ◽  
Architectural Proteins ◽  
Architectural Protein

Architectural proteins alter the shape of DNA, often by distorting the double helix and introducing sharp kinks that relieve strain in tightly-bent DNA structures. Here we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from E. coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

scholarly journals Designed architectural proteins that tune DNA looping in bacteria

2021 ◽  
Author(s):  
David H Tse ◽  
Nicole A Becker ◽  
Robert T Young ◽  
Wilma K Olson ◽  
Justin P Peters ◽  
...  
Keyword(s):  
Protein Binding ◽  
Double Helix ◽  
Dna Bending ◽  
Lac Repressor ◽  
Dna Looping ◽  
Transcription Activator ◽  
Dna Loops ◽  
Bent Dna ◽  
Architectural Proteins ◽  
Architectural Protein

Abstract Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

scholarly journals Structural insights into the role of architectural proteins in DNA looping deduced from computer simulations

2013 ◽  
Vol 41 (2) ◽  
pp. 559-564 ◽  
Author(s):  
Wilma K. Olson ◽  
Michael A. Grosner ◽  
Luke Czapla ◽  
David Swigon
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Genomic Sequence ◽  
Specific Binding ◽  
Double Helix ◽  
Lac Repressor ◽  
Dna Looping ◽  
Bacterial Gene ◽  
Architectural Proteins ◽  
Dna Double Helix

Bacterial gene expression is regulated by DNA elements that often lie far apart along the genomic sequence, but come close together during genetic processing. The intervening residues form loops, which are organized by the binding of various proteins. For example, the Escherichia coli Lac repressor protein binds DNA operators, separated by 92 or 401 bp, and suppresses the formation of gene products involved in the metabolism of lactose. The system also includes several highly abundant architectural proteins, such as the histone-like (heat-unstable) HU protein, which severely deform the double helix upon binding. In order to gain a better understanding of how the naturally stiff DNA double helix forms the short loops detected in vivo, we have developed new computational methods to study the effects of various non-specific binding proteins on the three-dimensional configurational properties of DNA sequences. The present article surveys the approach that we use to generate ensembles of spatially constrained protein-decorated DNA structures (minicircles and Lac repressor-mediated loops) and presents some of the insights gained from the correspondence between computation and experiment about the potential contributions of architectural and regulatory proteins to DNA looping and gene expression.

scholarly journals Characterization of gene repression by designed transcription activator-like effector dimer proteins

2020 ◽  
Author(s):  
NA Becker ◽  
JP Peters ◽  
TL Schwab ◽  
WJ Phillips ◽  
JP Wallace ◽  
...  
Keyword(s):  
Thermodynamic Model ◽  
Regulation Of Gene Expression ◽  
Fundamental Property ◽  
Lac Repressor ◽  
Dna Looping ◽  
Gene Repression ◽  
Length Variation ◽  
Transcription Activator ◽  
E Coli ◽  
Artificial Dna

AbstractGene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the E. coli lac operon switch, where DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed Transcription Activator-Like Effector Dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system.Statement of SignificanceWe are designing and testing in living bacteria artificial DNA looping proteins engineered based on principles learned from studies of the E. coli lac repressor. The engineered proteins are based on artificial dimers of Transcription Activator-Like Effector (TALE) proteins that have programmable DNA binding specificities. The current work is the first to create unique DNA repression loops using this approach. Systematic study of repression as a function of loop size, with data fitting to a thermodynamic model, now allows this system to be compared in detail with lac repressor loops, and relevant biophysical parameters to be estimated. This approach has implications for the artificial regulation of gene expression.

scholarly journals Selective DNA bending by a variety of bZIP proteins.

1993 ◽  
Vol 13 (9) ◽  
pp. 5479-5489 ◽  
Author(s):  
T K Kerppola ◽  
T Curran
Keyword(s):  
Dna Bending ◽  
Major Groove ◽  
Bent Dna ◽  
Dna Structures ◽  
Protein Dimers ◽  
Bzip Proteins ◽  
Related Proteins ◽  
Combinatorial Interactions ◽  
Bend Angles ◽  
Bzip Family

We have investigated DNA bending by bZIP family proteins that can bind to the AP-1 site. DNA bending is widespread, although not universal, among members of this family. Different bZIP protein dimers induced distinct DNA bends. The DNA bend angles ranged from virtually 0 to greater than 40 degrees as measured by phasing analysis and were oriented toward both the major and the minor grooves at the center of the AP-1 site. The DNA bends induced by the various heterodimeric complexes suggested that each component of the complex induced an independent DNA bend as previously shown for Fos and Jun. The Fos-related proteins Fra1 and Fra2 bent DNA in the same orientation as Fos but induced smaller DNA bend angles. ATF2 also bent DNA toward the minor groove in heterodimers formed with Fos, Fra2, and Jun. CREB and ATF1, which favor binding to the CRE site, did not induce significant DNA bending. Zta, which is a divergent member of the bZIP family, bent DNA toward the major groove. A variety of DNA structures can therefore be induced at the AP-1 site through combinatorial interactions between different bZIP family proteins. This diversity of DNA structures may contribute to regulatory specificity among the plethora of proteins that can bind to the AP-1 site.

scholarly journals A major role for Eco1 in regulating cohesin-mediated mitotic chromosome folding

2019 ◽  
Author(s):  
Lise Dauban ◽  
Rémi Montagne ◽  
Agnès Thierry ◽  
Luciana Lazar-Stefanita ◽  
Olivier Gadal ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Dna Looping ◽  
Loop Formation ◽  
Dna Loops ◽  
Sister Chromatids ◽  
Topologically Associating Domains ◽  
Dna Loop ◽  
Main Barrier ◽  
Structural Maintenance Of Chromosomes ◽  
Mitotic Chromatin

AbstractUnderstanding how chromatin organizes spatially into chromatid and how sister chromatids are maintained together during mitosis is of fundamental importance in chromosome biology. Cohesin, a member of the Structural Maintenance of Chromosomes (SMC) complex family, holds sister chromatids together 1–3 and promotes long-range intra-chromatid DNA looping 4,5. These cohesin-mediated DNA loops are important for both higher-order mitotic chromatin compaction6,7 and, in some organisms, compartmentalization of chromosomes during interphase into topologically associating domains (TADs) 8,9. Our understanding of the mechanism(s) by which cohesin generates large DNA loops remains incomplete. It involves a combination of molecular partners and active expansion/extrusion of DNA loops. Here we dissect the roles on loop formation of three partners of the cohesin complex: Pds5 10, Wpl1 11 and Eco1 acetylase 12, during yeast mitosis. We identify a new function for Eco1 in negatively regulating cohesin translocase activity, which powers loop extrusion. In the absence of negative regulation, the main barrier to DNA loop expansion appears to be the centromere. Those results provide new insights on the mechanisms regulating cohesin dependent DNA looping.

scholarly journals Intra-protein binding peptide fragments have specific and intrinsic sequence patterns

2017 ◽  
Author(s):  
Yuhong Wang ◽  
Junzhou Huang ◽  
Wei Li ◽  
Sheng Wang ◽  
Chuanfan Ding
Keyword(s):  
Protein Binding ◽  
Learning Algorithm ◽  
Double Helix ◽  
Peptide Fragments ◽  
Feed Forward Neural Network ◽  
Binding Peptide ◽  
Protein Protein Interaction ◽  
Deep Learning Algorithm ◽  
Sequence Patterns ◽  
Dna Double Helix

AbstractThe key finding in the DNA double helix model is the specific pairing or binding between nucleotides A-T and C-G, and the pairing rules are the molecule basis of genetic code. Unfortunately, no such rules have been discovered for proteins. Here we show that similar rules and intrinsic sequence patterns between intra-protein binding peptide fragments do exist, and they can be extracted using a deep learning algorithm. Multi-millions of binding and non-binding peptide fragments from currently available protein X-ray structures are classified with an accuracy of up to 93%. This discovery has the potential in helping solve protein folding and protein-protein interaction problems, two open and fundamental problems in molecular biology.One Sentence SummaryClassification of binding and non-binding intra-protein peptide fragments using feed-forward neural network

The unique structure of A-tracts and intrinsic DNA bending

2009 ◽  
Vol 42 (1) ◽  
pp. 41-81 ◽  
Author(s):  
Tali E. Haran ◽  
Udayan Mohanty
Keyword(s):  
Current Knowledge ◽  
Double Helix ◽  
Dna Bending ◽  
Helical Axis ◽  
Base Pairs ◽  
Regulatory Regions ◽  
Unique Structure ◽  
Distinct Structure ◽  
Global Curvature ◽  
Dna Double Helix

AbstractShort runs of adenines are a ubiquitous DNA element in regulatory regions of many organisms. When runs of 4–6 adenine base pairs (‘A-tracts’) are repeated with the helical periodicity, they give rise to global curvature of the DNA double helix, which can be macroscopically characterized by anomalously slow migration on polyacrylamide gels. The molecular structure of these DNA tracts is unusual and distinct from that of canonical B-DNA. We review here our current knowledge about the molecular details of A-tract structure and its interaction with sequences flanking them of either side and with the environment. Various molecular models were proposed to describe A-tract structure and how it causes global deflection of the DNA helical axis. We review old and recent findings that enable us to amalgamate the various findings to one model that conforms to the experimental data. Sequences containing phased repeats of A-tracts have from the very beginning been synonymous with global intrinsic DNA bending. In this review, we show that very often it is the unique structure of A-tracts that is at the basis of their widespread occurrence in regulatory regions of many organisms. Thus, the biological importance of A-tracts may often be residing in their distinct structure rather than in the global curvature that they induce on sequences containing them.

A Model for Highly Strained DNA in a Cavity

2011 ◽  
Author(s):  
Andrew D. Hirsh ◽  
Todd D. Lillian ◽  
N. C. Perkins
Keyword(s):  
Small Volume ◽  
Biological Function ◽  
Double Helix ◽  
Dna Bending ◽  
Elastic Rod ◽  
Viral Capsids ◽  
Elastic Rod Model ◽  
Highly Strained ◽  
Single Dna Molecule

A single DNA molecule is a long and flexible biopolymer that contains the genetic code. Building upon the discovery of the iconic double helix over 50 years ago, subsequent studies have emphasized how its biological function is related to the mechanical properties of the molecule. A remarkable system which high-lights the role of DNA bending and twisting is the packing and ejection of DNA into and from viral capsids. A recent 3D reconstruction of bacteriophage φ29 reveals a novel toroidal structure thought to be 30–40 bp of highly bent/twisted DNA contained in a small cavity below the capsid. Here, we extend an elastic rod model for DNA to enable simulation of the toroid as it is compacted and subsequently ejected from a small volume. We compute biologically-realistic forces required to form the toroid and predict ejection times of several nanoseconds.

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