Transcription Kinetics in the Coronavirus Life Cycle

Coronaviruses utilize a positive-sense single-strand RNA, functioning simultaneously as mRNA and the genome. An RNA-dependent RNA polymerase (RdRP) plays a dual role in transcribing genes and replicating the genome, making RdRP a critical target in therapies against coronaviruses. This review explores recent advancements in understanding the coronavirus transcription machinery, discusses it within virus infection context, and incorporates kinetic considerations on RdRP activity. We also address steric limitations in coronavirus replication, particularly during early infection phases, and outline hypothesis regarding translation–transcription conflicts, postulating the existence of mechanisms that resolve these issues. In cells infected by coronaviruses, abundant structural proteins are synthesized from subgenomic RNA fragments (sgRNAs) produced via discontinuous transcription. During elongation, RdRP can skip large sections of the viral genome, resulting in the creation of shorter sgRNAs that reflects the stoichiometry of viral structural proteins. Although the precise mechanism of discontinuous transcription remains unknown, we discuss recent hypotheses involving long-distance RNA–RNA interactions, helicase-mediated RdRP backtracking, dissociation and reassociation of RdRP, and RdRP dimerization

A beginners guide to Sf9 and Sf21 insect cell line culture and troubleshooting

Recombinant proteins are not only a crucial research tool but are also widely implemented in biomedicine. There are a number of expression systems used for recombinant protein production. Among them Spodoptera frugiperda (Sf) insect cell system is a powerful tool for multiprotein expression. Most commonly used are Sf9 and Sf21 cell lines due to their cost-effectiveness and availability. While a collection of protocols describing the Sf cell lines culturing is available, we have found them incomplete and their adaptation to real laboratory conditions challenging. Here we created a user-friendly hands-on protocol suitable for beginners. Our work combines the efforts of three independent laboratories which culture Sf cells, two labs with long experience, and one which recently successfully set up this system from scratch. We propose novel tricks and tips that allow for culturing of healthy Sf cells, and high protein yield production. Besides catering for beginners, our protocol can serve as a troubleshooting guide for more experienced researchers. We believe that this work is useful for biochemistry all the way to biomedical laboratories. Starting with an exhaustive description of Sf cell lines, through baculovirus expression vector system characteristic, this publication is a protocol, troubleshooting guide and compendium in one

The BRAHMA-associated SWI/SNF chromatin remodeling complex controls Arabidopsis seed quality and physiology

The SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex is involved in various aspects of plant development and stress responses. Here, we investigated the role of BRM (BRAHMA), a core catalytic subunit of the SWI/SNF complex, in Arabidopsis thaliana seed biology. brm-3 seeds exhibited enlarged size, reduced yield, increased longevity, and enhanced secondary dormancy, but did not show changes in primary dormancy or salt tolerance. Some of these phenotypes depended on the expression of DOG1, a key regulator of seed dormancy, as they were restored in the brm-3 dog1-4 double mutant. Transcriptomic and metabolomic analyses revealed that BRM and DOG1 synergistically modulate the expression of numerous genes. Some of the changes observed in the brm-3 mutant, including increased glutathione levels, depended on a functional DOG1. We demonstrated that the BRM-containing chromatin remodeling complex directly controls secondary dormancy through DOG1 by binding and remodeling its 3′ region, where the promoter of the long noncoding RNA asDOG1 is located. Our results suggest that BRM and DOG1 cooperate to control seed physiological properties and that BRM regulates DOG1 expression through asDOG1. This study reveals chromatin remodeling at the DOG1 locus as a molecular mechanism controlling the interplay between seed viability and dormancy

AlkA Glycosylase and AlkB Dioxygenase Constitute an Effective Protective System for Endogenously Arising Acrolein

Acrolein (ACR) is a ubiquitous environmental pollutant but also formed endogenously as a metabolite in oxidative stress conditions. Its adduct to adenine 1,N6-α-hydroxypropanoadenine (HPA) is a mutagenic lesion effectively repaired by the AlkB dioxygenase. Here, we provide in vivo, in vitro, and in silico evidence that it is also the substrate for the AlkA glycosylase. We studied the role of AlkA and AlkB in E. coli cells under conditions of induced adaptive response. Both alkA and alkB defective strains were not more sensitive to exogenous ACR than the wild type was. To simulate endogenously arising adducts, we used acrolein-modified plasmids, allowing monitoring of all kinds of substitutions originating from the acrolein modification of adenine. Both the AlkA and AlkB proteins were engaged in alleviating HPA-induced mutagenesis. Moreover, HPA was effectively repaired by AlkA and AlkB in vivo, even without induction of adaptive response. These findings suggest that the main contribution to acrolein mutagenicity comes from its endogenous sources, whereas AlkA and AlkB can play an additional role in controlling the level of DNA adducts of endogenous origin. Acrolein does not induce the adaptive response. HPA contains an asymmetric carbon atom in the hydroxypropano ring and exists as two stereoisomers. AlkA excises both of them in vitro. Molecular modelling demonstrated how dsDNA carrying both HPA stereoisomers could be properly bound at the AlkA catalytic centre. So, in contrast to the reaction catalyzed by AlkB, the HPA repair by AlkA is not expected to be stereoselective

Ribosomes translocation into the spore of Bacillus subtilis is highly organised and requires peptidoglycan rearrangements

In the spore-forming bacterium Bacillus subtilis transcription and translation are uncoupled and the translational machinery is located at the cell poles. During sporulation, the cell undergoes morphological changes including asymmetric division and chromosome translocation into the forespore. However, the fate of translational machinery during sporulation has not been described. Here, using microscopy and mass spectrometry, we show the localisation of ribosomes during sporulation in wild type and mutant Bacillus subtilis. We demonstrate that ribosomes are associated with the asymmetric septum, a functionally important organelle in the cell's developmental control, and that SpoIIDMP-driven peptidoglycan rearrangement is crucial for ribosomes packing into the forespore. We also show that the SpoIIIA-SpoIIQ 'feeding-tube' channel is not required for ribosome translocation. Our results demonstrate that translation and translational machinery are temporally and spatially organised in B. subtilis during sporulation and that the forespore 'inherits' ribosomes from the mother cell. We propose that the movement of ribosomes in the cell may be mediated by the bacterial homologs of cytoskeletal proteins and that the cues for asymmetric division localisation may be translation-dependent. We anticipate our findings to elicit more sophisticated structural and mechanistic studies of ribosome organisation during bacterial cell development

CRISPRi screen identifies FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa

With the rise of antibiotic-resistant bacteria, non-antibiotic therapies like gallium gain increasing attention. Intravenous gallium nitrate is under Phase II clinical trials to treat chronic Pseudomonas aeruginosa infections in cystic fibrosis patients. However, its clinical efficacy is constrained by the achievable peak concentration in human tissue. To address this limitation, we apply a genome-wide CRISPR interference approach (CRISPRi-seq) to identify potential synergistic targets with gallium. We classify the essential genes by response time and growth reduction, pinpointing the most vulnerable therapeutic targets in this species. In addition, we identify a highly conserved gene, fprB, encoding a ferredoxin-NADP⁺ reductase, whose deletion sensitizes P. aeruginosa to gallium, lowering its MIC by 32-fold and shifting mode of action from bacteriostatic to bactericidal. Further investigation reveals that FprB plays a critical role in modulating oxidative stress induced by gallium, via control of iron homeostasis and reactive oxygen species accumulation. Deleting fprB enhances gallium’s efficacy against biofilm formation and improves outcomes in a murine lung infection model of P. aeruginosa, suggesting FprB is a promising drug target in combination with gallium. Overall, our data show CRISPRi-seq as a powerful tool for systematic genetic analysis of P. aeruginosa, advancing the identification of novel therapeutic targets

Genome sequences of polar Carnobacterium maltaromaticum strains 2857 and 2862 with genes for glycerol and 1,2-propanediol pathways

We report genome sequences of two polar Carnobacterium maltaromaticum strains: 2857 (draft, 3.54 Mb, 34.4% GC) and 2862 (complete, 3.61 Mb, 34.6% GC, five plasmids). Sequencing used Illumina (both) and Nanopore (2862). Genome analysis revealed genes for glycerol conversion to 1,2-propanediol, suggesting potential for sustainable bioprocessing in cold environments

SWI/SNF-type complexes–transcription factor interplay: a key regulatory interaction

ATP-dependent switch/sucrose nonfermenting-type chromatin remodeling complexes (SWI/SNF CRCs) are multiprotein machineries altering chromatin structure, thus controlling the accessibility of genomic DNA to various regulatory proteins including transcription factors (TFs). SWI/SNF CRCs are highly evolutionarily conserved among eukaryotes. There are three main subtypes of SWI/SNF CRCs: canonical (cBAF), polybromo (pBAF), and noncanonical (ncBAF) in humans and their functional Arabidopsis counterparts SYD-associated SWI/SNF (SAS), MINU-associated SWI/SNF (MAS), and BRAHMA (BRM)-associated SWI/SNF (BAS). Here, we highlight the importance of interplay between SWI/SNF CRCs and TFs in human and Arabidopsis and summarize recent advances demonstrating their role in controlling important regulatory processes. We discuss possible mechanisms involved in TFs and SWI/SNF CRCs-dependent transcriptional control of gene expression. We indicate that Arabidopsis may serve as a valuable model for the identification of evolutionarily conserved SWI/SNF–TF interactions and postulate that further exploration of the TFs and SWI/SNF CRCs-interplay, especially in the context of the role of particular SWI/SNF CRC subtypes, TF type, as well as cell/tissue and conditions, among others, will help address important questions related to the specificity of SWI/SNF–TF interactions and the sequence of events occurring on their target genes

Tandem mass tag-based proteomic analysis of granulosa and theca interna cells of the porcine ovarian follicle following in vitro treatment with vitamin D3 and insulin alone or in combination

This study was performed to investigate the proteomic basis underlying the interaction between vitamin D3 (VD) and insulin (I) within ovarian follicle using the pig as a model. Porcine antral follicles were incubated in vitro for 12 h with VD alone and I alone or in combination (VD + I) or with no treatment as the control (C). In total, 7690 and 7467 proteins were identified in the granulosa and theca interna compartments, respectively. Comparative proteomic analysis revealed 97 differentially abundant proteins (DAPs) within the granulosa layer and 11 DAPs within the theca interna layer. In the granulosa compartment, VD affected proteome leading to the promotion of cell proliferation, whereas I influenced mainly proteins related to cellular adhesion. The VD + I treatment induced granulosa cell proliferation probably via the DAPs involved in DNA synthesis and the cell cycle regulation. In the theca interna layer, VD alone or in co-treatment with I affected DAPs associated with cholesterol transport and lipid and steroid metabolic processes that was further confirmed by diminished lipid droplet accumulation