Psoralen mapping reveals a bacterial genome supercoiling landscape dominated by transcription.

DNA supercoiling is a key regulator of all DNA metabolic processes including replication, transcription, and recombination, yet a reliable genomic assay for supercoiling is lacking. Here, we present a robust and flexible method (Psora-seq) to measure whole-genome supercoiling at high resolution. Using this tool in Escherichia coli, we observe a supercoiling landscape that is well correlated to transcription. Supercoiling twin-domains generated by RNA polymerase complexes span 25 kb in each direction - an order of magnitude farther than previous measurements in any organism. Thus, ribosomal and many other highly expressed genes strongly affect the topology of about 40 neighboring genes each, creating highly integrated gene circuits. Genomic patterns of supercoiling revealed by Psora-seq could be aptly predicted from modeling based on gene expression levels alone, indicating that transcription is the major determinant of chromosome supercoiling. Large-scale supercoiling patterns were highly symmetrical between left and right chromosome arms (replichores), indicating that DNA replication also strongly influences supercoiling. Skew in the axis of symmetry from the natural ori-ter axis supports previous indications that the rightward replication fork is delayed several minutes after initiation. Implications of supercoiling on DNA replication and chromosome domain structure are discussed.

Methylxanthines as Potential Inhibitor of SARS-CoV-2: an In Silico Approach.

The aim of the present study was to test the binding affinity of methylxanthines (caffeine/theine, methylxanthine, theobromine, theophylline and xanthine) to three potential target proteins namely Spike protein (6LZG), main protease (6LU7) and nucleocapsid protein N-terminal RNA binding domain (6M3M) of SARS-CoV-2. Proteins and ligand were generated using AutoDock 1.5.6 software. Binding affinity of methylxanthines with SARS-CoV-2 target proteins was determined using Autodock Vina. MD simulation of the best interacting complexes was performed using GROMACS 2018.3 (in duplicate) and Desmond program version 2.0 (academic version) (in triplicate) to study the stabile interaction of protein-ligand complexes. Among the selected methylxanthines, theophylline showed the best binding affinity with all the three targets of SARS-CoV-2 (6LZG - 5.7 kcal mol-1, 6LU7 - 6.5 kcal mol-1, 6M3M - 5.8 kcal mol-1). MD simulation results of 100 ns (in triplicate) showed that theophylline is stable in the binding pockets of all the selected SARS-CoV-2 proteins. Moreover, methylxanthines are safer and less toxic as shown by high LD50 value with Protox II software as compared to drug chloroquine. This research supports the use of methylxanthines as a SARS-CoV-2 inhibitor. It also lays the groundwork for future studies and could aid in the development of a treatment for SARS-CoV-2 and related viral infections. Supplementary Information: The online version contains supplementary material available at 10.1007/s40495-021-00276-3.

Liquid condensation of reprogramming factor KLF4 with DNA provides a mechanism for chromatin organization.

Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid-liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.

Saccharomyces cerevisiae ER membrane protein complex subunit 4 (EMC4) plays a crucial role in eIF2B-mediated translation regulation and survival under stress conditions.

BACKGROUND: Eukaryotic initiation factor 2B (eIF2B) initiates and regulates translation initiation in eukaryotes. eIF2B gene mutations cause leukoencephalopathy called vanishing white matter disease (VWM) in humans and slow growth (Slg-) and general control derepression (Gcd-) phenotypes in Saccharomyces cerevisiae. RESULTS: To suppress eIF2B mutations, S. cerevisiae genomic DNA library was constructed in high-copy vector (YEp24) and transformed into eIF2B mutant S. cerevisiae strains. The library was screened for wild-type genes rescuing S. cerevisiae (Slg-) and (Gcd-) phenotypes. A genomic clone, Suppressor-I (Sup-I), rescued S. cerevisiae Slg- and Gcd- phenotypes (gcd7-201 gcn2∆). The YEp24/Sup-I construct contained truncated TAN1, full length EMC4, full length YGL230C, and truncated SAP4 genes. Full length EMC4 (chaperone protein) gene was sub-cloned into pEG (KG) yeast expression vector and overexpressed in gcd7-201 gcn2∆ strain which suppressed the Slg- and Gcd- phenotype. A GST-Emc4 fusion protein of 47 kDa was detected by western blotting using α-GST antibodies. Suppression was specific to gcd7-201 gcn2∆ mutation in eIF2Bß and Gcd1-502 gcn2∆ in eIF2Bγ subunit. Emc4p overexpression also protected the wild type and mutant (gcd7-201 gcn2∆, GCD7 gcn2∆, and GCD7 GCN2∆) strains from H2O2, ethanol, and caffeine stress. CONCLUSIONS: Our results suggest that Emc4p is involved in eIF2B-mediated translational regulation under stress and could provide an amenable tool to understand the eIF2B-mediated defects.

Role of Saccharomyces cerevisiae TAN1 (tRNA acetyltransferase) in eukaryotic initiation factor 2B (eIF2B)-mediated translation control and stress response.

Eukaryotic initiation factor 2B (eIF2B) controls the first step of translation by catalyzing guanine nucleotide exchange on eukaryotic initiation factor 2 (eIF2). Mutations in the genes encoding eIF2B subunits inhibit the nucleotide exchange and eventually slow down the process of translation, causing vanishing white matter disease. We constructed a Saccharomyces cerevisiae genomic DNA library in YEp24 vector and screened it for the identification of extragenic suppressors of eIF2B mutations, corresponding to human eIF2B mutations. We found a suppressor-II (Sup-II) genomic clone, as suppressor of eIF2Bß (gcd7-201) mutation. Identification of Sup-II reveals the presence of truncated SEC15, full-length TAN1 (tRNA acetyltransferase), full-length EMC4, full-length YGL230C (putative protein) and truncated SAP4 genes. Full-length TAN1 (tRNA acetyltransferase) gene, subcloned into pEG(KG) vector and overexpressed in gcd7-201 gcn2∆ strain, suppresses the slow-growth (Slg-) and general control derepression (Gcd-) phenotype of gcd7-201 gcn2∆ mutation, but YGL230C did not show any effect. A GST-Tan1p fusion protein of 60 kDa was detected by western blotting using α-GST antibodies. Interestingly, Tan1p overexpression also suppresses the temperature-sensitive (Ts-), Slg- and Gcd- phenotype of eIF2Bγ (gcd1-502) mutant. Role of Tan1p protein in eIF2B-mediated translation regulation was also studied. Results revealed that Tan1p overexpression confers resistance to GCD7 GCN2, gcd7-201 gcn2∆, GCD7 gcn2∆ growth defect under ethanol, H2O2 and caffeine stress. No resistance to DMSO-, NaCl- and DTT-mediated growth defect upon GCD7 gcn2∆, GCD7 GCN2, gcd7-201 gcn2∆ was observed by overexpression of TAN1. Hence, we proposed that Tan1p is involved directly or indirectly in regulating eIF2B-mediated translation.