Differential roles of positive and negative supercoiling in organizing the E. coli genome

Nucleic Acids Research, Год журнала: 2023, Номер 52(2), С. 724 - 737

Опубликована: Дек. 5, 2023

Abstract This study aims to explore whether and how positive negative supercoiling contribute the three-dimensional (3D) organization of bacterial genome. We used recently published Escherichia coli GapR ChIP-seq TopoI (also called EcTopoI-seq) data, which marks sites, respectively, correlates with spatial contact maps obtained from chromosome conformation capture sequencing (Hi-C 5C). find that supercoiled chromosomal loci have overall higher Hi-C frequencies than sites are not supercoiled. Surprisingly, corresponds supercoiling. Additionally, positive, but negative, could be identified data high accuracy. further majority supercoils coincide highly active transcription units, a minor group likely associated replication other genomic processes. Our results show both enhance contact, playing larger role in bringing closer space. Based on our results, we propose new physical models E. is organized by supercoils.

Язык: Английский

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DNA supercoiling modulates eukaryotic transcription in a gene-orientation dependent manner

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 3, 2025

Summary Transcription introduces torsional stress in the DNA fiber causing it to transition from a relaxed supercoiled state that can propagate across several kilobases and modulate binding activity of DNA-associated proteins. As result, transcription at one locus has potential impact nearby events. In this study, we asked how supercoiling affects histone modifications neighboring genes multicellular eukaryote Caenorhabditis elegans . We acutely depleted two major topoisomerases measured nascent by Global Run-on sequencing (GRO-seq), RNA Polymerase II occupancy ChIP-seq, gene expression RNA-seq four transcription-associated Cut & Tag. Depletion I led genome-wide changes dynamics, with minor disruptions modification landscape. Our results showed C. topoisomerase is required for elongation partially redundant II. Analysis respect suggest negative promotes divergent neighbor positive suppresses convergent genes. Additionally, depletion caused coordinated pairs, suggesting drives their synchronized expression. Conversely, was disrupted, excessive inhibits transcription. Overall, our data supports model which generated site propagates along eukaryotic chromatin fiber, influencing an orientation-dependent manner.

Язык: Английский

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Towards a Better Control of Engineered Circuit Transcription in Bacterial Genomes

Applied Microbiology, Год журнала: 2025, Номер 5(1), С. 17 - 17

Опубликована: Фев. 7, 2025

The transcription of genes and engineered circuits can deeply vary when inserted into different genomic loci. This unpredictable performance, termed context sensitivity, complicates strain development. Although the causes mechanisms sensitivity are emerging, it is poorly known how to engineer synthetic pathways isolated from it. Using tools biology for designing inserting various reporter cassettes in Escherichia coli genome RT-qPCR directly measuring gene transcription, we first surveyed landscape at 214 positions cells grown glucose or glycerol. results show deep variations cassette with respect position (up 160-fold) growth condition a 30-fold). We then demonstrated that this position-dependent variability strongly reduced insulated an artificial protein-bound DNA loop. Finally, measured two loop-insulated positions. depends on relative orientation genes, promoter strength, positive supercoiling. present model suggesting looping important cause be used better controlling circuits.

Язык: Английский

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High-Resolution Genome-Wide Maps Reveal Widespread Presence of Torsional Insulation

Опубликована: Фев. 18, 2025

Torsional stress in chromatin plays a fundamental role cellular functions, influencing key processes such as transcription, replication, and organization. Transcription other may generate be regulated by torsional stress. In the genome, interplay of these creates complicated patterns both positive (+) negative (-) torsion. However, challenge generating an accurate torsion map is determining zero-torsion baseline signal, which conflated with accessibility. Here, we introduce high-resolution method based on intercalator trimethylpsoralen (TMP) to address this challenge. We describe establish while preserving state genome S. cerevisiae . This approach enables mapping accessibility cell. Our analysis shows transcription-generated domains consistent twin-supercoiled-domain model transcription suggests for recruiting topoisomerases regulating 3D architecture via cohesin. Significantly, reveal that insulator sequence-specific factors decouple between divergent promoters, whereas spreads promoters lacking factors, suggesting serves regulatory mechanism regions. Although insulators are known gene expression, our finding provides physical explanation how decoupling occur. new potential path forward using TMP measure without confounding contribution chromatin.

Язык: Английский

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Title: Engineering Plasmids with Synthetic Origins of Replication

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Фев. 21, 2025

Abstract Plasmids remain by far the most common medium for delivering engineered DNA to microorganisms. However, reliance on natural plasmid replication mechanisms limits their tunability, compatibility, and modularity. Here we refactor pMB1 origin create plasmids with customizable copy numbers tuning refactored components. We then compatible origins that use synthetic RNA regulators implement independent control. further demonstrate of (SynORI) can be modularly respond various signals, allowing multiplexed copy-based reporting environmental signals. Lastly, a library 6 orthogonal SynORI is created co-maintained in E. coli week. This work establishes feasibility creating serve as new biotechnology biology.

Язык: Английский

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