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

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.

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Designed architectural proteins that tune DNA looping in bacteria

Nucleic Acids Research ◽

10.1093/nar/gkab759 ◽

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.

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Designed architectural proteins that tune DNA looping in bacteria

10.1101/2021.06.30.450240 ◽

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.

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Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization

Nucleic Acids Research ◽

10.1093/nar/gkz645 ◽

2019 ◽

Vol 47 (18) ◽

pp. e109-e109

Author(s):

Eliraz Greenberg ◽

Hodaya Hochberg-Laufer ◽

Shalev Blanga ◽

Noa Kinor ◽

Yaron Shav-Tal

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Dna Sequences ◽

Plasmid Dna ◽

Double Helix ◽

Rna Molecules ◽

Rna Fish ◽

Dna Double Helix ◽

Intracellular Detection

Abstract Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.

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Comparison of the Global Genomic and Transcription-Coupled Repair Rates of Different Lesions in Human Cells

Zeitschrift für Naturforschung C ◽

10.1515/znc-2004-5-628 ◽

2004 ◽

Vol 59 (5-6) ◽

pp. 445-453 ◽

Cited By ~ 5

Author(s):

Boyko Atanassov ◽

Aneliya Velkova ◽

Emil Mladenov ◽

Boyka Anachkova ◽

George Russev

Keyword(s):

Dna Sequences ◽

Chemical Nature ◽

Excision Repair ◽

Uv Light ◽

Double Helix ◽

Human Cells ◽

The Other ◽

Specific Sequence ◽

Dna Double Helix ◽

Transcription Coupled Repair

There are two subclasses of nucleotide excision repair (NER). One is the global genomic repair (GGR) which removes lesions throughout the genome regardless of whether any specific sequence is transcribed or not. The other is the transcription-coupled repair (TCR), which removes lesions only from the transcribed DNA sequences. There are data that GGR rates depend on the chemical nature of the lesions in a manner that the lesions inflicting larger distortion on the DNA double helix are repaired at higher rate. It is not known whether the TCR repair rates depend on the type of lesions and in what way. To address this question human cells were transfected with pEGFP and pEYFP plasmids treated with UV light, cis-diamminedichloroplatinum(II) (cisplatin) and angelicin and 24 h later the restored fluorescence was measured and used to calculate the respective NER rates. In a parallel series of experiments the same plasmids were incubated in repair-competent protein extracts to determine GGR rates in the absence of transcription. From the two sets of data, the TCR rates were calculated. We found out that cisplatin, UV light and angelicin lesions were repaired by GGR with different efficiency, which corresponded to the degree of DNA helix distortion induced by these agents. On the other hand the three lesions were repaired by TCR at very similar rates which showed that TCR efficiency was not directly connected with the chemical nature of the lesions.

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Conformational variability of DNA double helix

VEGETABLE CROPS OF RUSSIA ◽

10.18619/2072-9146-2020-6-51-57 ◽

2020 ◽

pp. 51-57

Author(s):

Yu. V. Chesnokov

Keyword(s):

Gene Expression ◽

Metal Ions ◽

Double Helix ◽

General Structure ◽

Regulation Of Gene Expression ◽

Nucleotide Composition ◽

Conformational Variability ◽

Living Organisms ◽

Dna Double Helix

Deoxyribonucleic acid (DNA) is one of the main carriers of hereditary information. The structural physicochemical information of DNA ultimately determines the structure and functioning of all living organisms. In DNA, various mutational events accumulate and recombination events occur, which lead to the variability of organisms and are subject to both natural and artificial selection. The interaction "genotype-environment" inherent in all living organisms is also characteristic of DNA, which is located in the intracellular and intranuclear physicochemical environment of water molecules, sugars, metal ions, pH, nucleotides and other components. The establishment and study of the physicochemical properties of native DNA contributes to not only understanding the mechanisms of the structure of the main hereditary biomolecule, but also to clarify their functioning, as well as interaction with other molecules at the molecular level. The discovery of various forms of double helices: A, Aʹ, B, α-Bʹ, β-Bʹ, C, Cʹ, Cʹʹ, D, E and Z suggests the idea of molecular genetic diversity existing at the DNA level and the establishment of their structural and functional features can lead to an understanding of the implementation of genetic information at the general biological level. The structure of natural DNA as a whole, apparently, does not depend on the sequence and nucleotide composition. For natural molecules - satellite DNA with repeats or DNA without repeats, the presence of only A-, B- and C-forms has been confirmed. The structure of DNA depends not only on temperature, but also on the nature of the cations present. The presence of a certain amount of metal ions in the medium can lead to the transition of the B-form of DNA to the Zform. The B ↔ Z transition modifies the general structure of DNA and, therefore, may be important for the regulation of gene expression. The study of the biological role of Z-DNA, possibly in the near future, will help to understand the mechanism of gene expression, primarily of an epigenetic nature, which has not yet been fully elucidated.

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The regulatory function of dIno80 correlates with its DNA binding activity

10.1101/519959 ◽

2019 ◽

Cited By ~ 1

Author(s):

S Jain ◽

J Maini ◽

A Narang ◽

S Maiti ◽

V Brahmachari

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Dna Sequences ◽

Specific Binding ◽

Binding Domain ◽

Regulatory Function ◽

Binding Potential ◽

Homeotic Genes ◽

Dna Binding Domain

ABSTRACTThe INO80 complex, including the Ino80 protein, forms a highly conserved canonical complex that remodels chromatin in the context of multiple cellular functions. TheDrosophilahomologue, dIno80, is involved in homeotic gene regulation during development as a canonical Pho-dIno80 complex. Previously, we found that dIno80 regulates homeotic genes by interacting with epigenetic regulators, such as polycomb and trithorax, suggesting the occurrence of non-canonical Ino80 complexes. Here using spectroscopic methods and gel retardation assays, we identified a set of consensus DNA sequences that DNA binding domain of dIno80 (DBINO) interacts with having differential affinity and high specificity. Testing these sequences in reporter assays, showed that this interaction can positively regulate transcription. These results suggest that, dIno80 has a sequence preference for interaction with DNA leading to transcriptional changes.SIGNIFICANCEThe chromatin remodeling proteins control gene expression by nucleosome sliding and exchange. They are known to function as multi-subunit complexes recruited to chromatin by transcription factors or histone modification readers. Here, we report a sequence specific binding potential for the chromatin remodeler, dIno80. We have carried outin vitrostudies with DNA binding domain of dIno80 to elucidate its sequence specific DNA binding. We have also showed that this binding can regulated reporter gene expression inDrosophilacells. Our results suggest a non-canonical role of Ino80 in transcriptional regulation.

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Exploring different sequence representations and classification methods for the prediction of nucleosome positioning

10.1101/482612 ◽

2018 ◽

Cited By ~ 1

Author(s):

Nikos Kostagiolas ◽

Nikiforos Pittaras ◽

Christoforos Nikolaou ◽

George Giannakopoulos

Keyword(s):

Gene Expression ◽

Dna Sequences ◽

Large Scale ◽

Genomic Sequence ◽

Critical Role ◽

Regulation Of Gene Expression ◽

Nucleosome Positioning ◽

Regulatory Elements ◽

Machine Learning Algorithms ◽

N Gram

Nucleosomes form the first level of DNA compaction and thus bear a critical role in the overall genome organization. At the same time, they modulate chromatin accessibility and, through a dynamic equilibrium with other DNA-binding proteins, may shape gene expression. A number of large-scale nucleosome positioning maps, obtained for various genomes, has compelled the importance of nucleosomes in the regulation of gene expression and has shown constraints in the relative positions of nucleosomes to be much stronger around regulatory elements (i.e. promoters, splice junctions and enhancers). At the same time, the great majority of nucleosome positions appears to be rather flexible. Various computational methods have in the past been used in order to capture the sequence determinants of nucleosome positioning but, as the extent to which DNA sequence preferences may guide nucleosome occupancy largely varies, this has proved to be rather difficult. In order to focus on highly specific sequence attributes, in this work we have analyzed two well-defined sets of nucleosome-occupied sites (NOS) and nucleosome-free-regions (NFR) from the genome of S. cerevisiae, with the use of textual representations. We employed 3 different genomic sequence representations (Hidden Markov Models, Bag-of-Words and N-gram Graphs) combined with a number of machine learning algorithms on the task of classifying genomic sequences as nucleosome-free (NFR) or nucleosome-occupied NOS (to be further amended based on updated results). We found that different approaches that involve the usage of different representations or algorithms can be more or less effective at predicting nucleosome positioning based on the textual data of the underlying genomic sequence. More interestingly, we show that N-gram Graphs, a sequence representation that takes into account both k-mer occurrences and relative positioning at various lengths scales is outperforming other methodologies and may thus be a choice of preference for the analysis of DNA sequences with subtle constraints.

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Improved Production of l-Threonine in Escherichia coli by Use of a DNA Scaffold System

Applied and Environmental Microbiology ◽

10.1128/aem.02578-12 ◽

2012 ◽

Vol 79 (3) ◽

pp. 774-782 ◽

Cited By ~ 49

Author(s):

Jun Hyoung Lee ◽

Suk-Chae Jung ◽

Le Minh Bui ◽

Kui Hyeon Kang ◽

Ji-Joon Song ◽

...

Keyword(s):

Escherichia Coli ◽

Zinc Finger Protein ◽

Specific Binding ◽

Double Helix ◽

Local Concentration ◽

Simple Diffusion ◽

Content Type ◽

Threonine Production ◽

Dna Double Helix ◽

Dna Scaffold

ABSTRACTDespite numerous approaches for the development ofl-threonine-producing strains, strain development is still hampered by the intrinsic inefficiency of metabolic reactions caused by simple diffusion and random collisions of enzymes and metabolites. A scaffold system, which can promote the proximity of metabolic enzymes and increase the local concentration of intermediates, was reported to be one of the most promising solutions. Here, we report an improvement inl-threonine production inEscherichia coliusing a DNA scaffold system, in which a zinc finger protein serves as an adapter for the site-specific binding of each enzyme involved inl-threonine production to a precisely ordered location on a DNA double helix to increase the proximity of enzymes and the local concentration of metabolites to maximize production. The optimized DNA scaffold system forl-threonine production significantly increased the efficiency of the threonine biosynthetic pathway inE. coli, substantially reducing the production time forl-threonine (by over 50%). In addition, this DNA scaffold system enhanced the growth rate of the host strain by reducing the intracellular concentration of toxic intermediates, such as homoserine. Our DNA scaffold system can be used as a platform technology for the construction and optimization of artificial metabolic pathways as well as for the production of many useful biomaterials.

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Coherent Domains of Transcription Coordinate Gene Expression During Bacterial Growth and Adaptation

Microorganisms ◽

10.3390/microorganisms7120694 ◽

2019 ◽

Vol 7 (12) ◽

pp. 694 ◽

Cited By ~ 4

Author(s):

Georgi Muskhelishvili ◽

Raphaël Forquet ◽

Sylvie Reverchon ◽

Sam Meyer ◽

William Nasser

Keyword(s):

Gene Expression ◽

Bacterial Growth ◽

Dna Sequences ◽

Thermodynamic Stability ◽

Spatial Organization ◽

Genomic Sequence ◽

Transcriptional Responses ◽

Sequence Organization ◽

Temporal Gene Expression ◽

Coordinated Expression

Recent studies strongly suggest that in bacteria, both the genomic pattern of DNA thermodynamic stability and the order of genes along the chromosomal origin-to-terminus axis are highly conserved and that this spatial organization plays a crucial role in coordinating genomic transcription. In this article, we explore the relationship between genomic sequence organization and transcription in the commensal bacterium Escherichia coli and the plant pathogen Dickeya. We argue that, while in E. coli the gradient of DNA thermodynamic stability and gene order along the origin-to-terminus axis represent major organizational features orchestrating temporal gene expression, the genomic sequence organization of Dickeya is more complex, demonstrating extended chromosomal domains of thermodynamically distinct DNA sequences eliciting specific transcriptional responses to various kinds of stress encountered during pathogenic growth. This feature of the Dickeya genome is likely an adaptation to the pathogenic lifestyle utilizing differences in genomic sequence organization for the selective expression of virulence traits. We propose that the coupling of DNA thermodynamic stability and genetic function provides a common organizational principle for the coordinated expression of genes during both normal and pathogenic bacterial growth.

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Comparative Analysis of Sequence Periodicity among Prokaryotic Genomes Points to Differences in Nucleoid Structure and a Relationship to Gene Expression

Journal of Bacteriology ◽

10.1128/jb.00149-10 ◽

2010 ◽

Vol 192 (14) ◽

pp. 3763-3772 ◽

Cited By ~ 12

Author(s):

Jan Mrázek

Keyword(s):

Dna Sequences ◽

Double Helix ◽

Integral Form ◽

Periodic Signal ◽

Dna Curvature ◽

Periodic Patterns ◽

Bent Dna ◽

Sequence Periodicity ◽

Prokaryotic Genomes ◽

Dna Double Helix

ABSTRACT Regular spacing of short runs of A or T nucleotides in DNA sequences with a period close to the helical period of the DNA double helix has been associated with intrinsic DNA bending and nucleosome positioning in eukaryotes. Analogous periodic signals were also observed in prokaryotic genomes. While the exact role of this periodicity in prokaryotes is not known, it has been proposed to facilitate the DNA packaging in the prokaryotic nucleoid and/or to promote negative or positive supercoiling. We developed a methodology for assessments of intragenomic heterogeneity of these periodic patterns and applied it in analysis of 1,025 prokaryotic chromosomes. This technique allows more detailed analysis of sequence periodicity than previous methods where sequence periodicity was assessed in an integral form across the whole chromosome. We found that most genomes have the periodic signal confined to several chromosomal segments while most of the chromosome lacks a strong sequence periodicity. Moreover, there are significant differences among different prokaryotes in both the intensity and persistency of sequence periodicity related to DNA curvature. We proffer that the prokaryotic nucleoid consists of relatively rigid sections stabilized by short intrinsically bent DNA segments and characterized by locally strong periodic patterns alternating with regions featuring a weak periodic signal, which presumably permits higher structural flexibility. This model applies to most bacteria and archaea. In genomes with an exceptionally persistent periodic signal, highly expressed genes tend to concentrate in aperiodic sections, suggesting that structural heterogeneity of the nucleoid is related to local differences in transcriptional activity.

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