RNA Polymerase Research Articles

Catching the glucocorticoid receptor in the act: Lessons from fluorescence fluctuation methods.

Technological innovation can drive scientific inquiry by allowing researchers to answer questions that were once out of reach. Eukaryotic mRNA synthesis was not so long ago thought of as a deterministic, sequential process in which transcriptional regulators and general transcription factors assemble in an orderly fashion into chromatin to, ultimately, activate RNA polymerase II. Advances in fluorescence microscopy techniques have revealed a much more complex scenario, wherein transcriptional regulators dynamically engage with chromatin in a more stochastic, probabilistic way. In this review, we will concentrate on what fluorescence fluctuation methods have taught us about the journey of transcription factors within live cells. Specifically, we summarized how these techniques have contributed to reshaping our understanding of the mechanism(s) of action of the glucocorticoid receptor, a ligand-regulated transcription factor involved in many physiological and pathological processes. This receptor regulates a variety of gene networks in a context-specific manner and its activity can be quickly and easily controlled by the addition of specific ligands. Thus, it is widely used as a model to study the mechanisms of transcription factors through live-cell imaging.

Customized design of host-independent T7 expression system (HITES) for a broad host range.

Efficient methods and universal DNA elements are eagerly required for the expression of proteins and the production of target chemicals in synthetic biology and metabolic engineering. This paper develops a customized-design approach by utilizing the host-independent T7 expression system (HITES), which facilitates the rational design and rapid construction of T7 expression systems. Firstly, the EiL (Upper-limit value of initial enzyme activity) value is discovered to play a pivotal factor in the successful construction of the T7 expression system, different host strains exhibit varying EiL values, and this study presents a method to measure the EiL values. Secondly, E. coli DH5α is chosen as the host strain, and it demonstrates that various strategies to modulate T7 RNA polymerase activity can efficiently construct the HITES T7 expression system in E. coli DH5α under the guidance of EiL. Lastly, the customized-design of HITES enables the efficient expression of sfGFP and D-MIase proteins across 13 host strains, guided by EiL values. This customized-design method of HITES offers a streamlined pathway for T7 system construction across a broad range of hosts and serves as an enabling tool for synthetic biology, metabolic engineering, and enzyme engineering.

Transcription Regulation of Flagellins: A Structural Perspective.

Bacterial flagella are complex molecular motors that are essential for locomotion and host colonization. They consist of 30 different proteins expressed in varying stoichiometries. Their assembly and function are governed by a hierarchical transcriptional regulatory network with multiple checkpoints primarily regulated by sigma factors. Expression of late flagellar genes requires the complete assembly of the flagellar basal body and hook. The extracellular segment of the flagellum, termed filament, is composed of self-assembling flagellin subunits encoded by the fliC gene and harbors potent antigenic epitopes. Structural studies have illuminated the molecular mechanisms underlying its assembly and its regulation at the transcription level. σ28, a key subunit of the RNA polymerase complex, binds to specific promoter sequences to initiate transcription of late flagellar genes, while its activity is controlled by the antisigma factor FlgM. This review summarizes current insights into the structural characterization of flagellins across various bacterial species, their transcription by σ28, and the structural mechanism controlling σ28 activity through FlgM. Additionally, we highlight the regulation of flagellin gene expression via transcription factors and their post-transcriptional regulation, providing a comprehensive overview of the intricate mechanisms that support bacterial motility and adaptation.

Aggressiveness and phylogenetic relationship of Fusarium oxysporum associated with crown and root rot in pyrethrum plants.

In Australia, pyrethrum (Tanacetum cinerariifolium) cultivation provides a significant portion of the global supply of natural insecticidal pyrethrins. However, crown and root rots, along with stunted plant growth and plant loss during winter, are significant issues affecting certain sites. Several isolates of the Fusarium oxysporum species complex (FOSC) have been identified as causal agents of crown and root rot in pyrethrum, highlighting these as key pathogens contributing to this decline. However, the genetic and pathogenic diversity of the FOSC impacting pyrethrum is unclear. This study isolated F. oxysporum consistently from symptomatic and asymptomatic field-grown pyrethrum plant tissues, identified through morphological and multigene phylogenetic analyses. Phylogenetic analyses of partial gene sequences of calmodulin (cmdA), RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1-α) and β-tubulin (tub2) resolved the placement of these isolates within the context of different published FOSC taxonomies and revealed notable genetic diversity among the isolates. Glasshouse experiments effectively reproduced the crown and root rot symptoms observed in field conditions, demonstrating a similar level of aggressiveness among F. oxysporum isolates from pyrethrum plants. The results indicate the importance of soil-borne disease management to reduce yield decline in pyrethrum fields and will help with the selection of aggressive isolates for resistance screening of pyrethrum varieties.

The S-Phase Arrest of Host Cells Caused by an Alpha-Herpesvirus Genome Replication Facilitates Viral Recruitment of RNA Polymerase II to Transcribe Viral Genes.

Herpesviruses rely on host RNA polymerae II (RNA Pol II) for their mRNA transcription, yet the mechanisms of which has been poorly defined, while certain herpesviruses can enhance viral gene transcription by altering the RNA Pol II location, modulating its phosphorylation, or directly interacting with RNA Pol II. However, the influence of herpesviruses on RNA Pol II transcription extends beyond these direct effects. Here, we present a novel mechanism by which the host cell cycle regulates viral gene transcription via RNA Pol II during infection by Anatid Herpesvirus 1 (AnHV-1), an avian alpha-herpesvirus. The results demonstrated that the formation of viral replication compartments (vRCs) and the subsequent recruitment of RNA pol II are positively correlated with AnHV-1 DNA synthesis. As viral DNA replication progresses, host cells are arrested in the S phase, which not only halts host gene transcription but also facilitates viral transcription. This cell cycle arrest in the S phase promotes viral DNA (vDNA) synthesis and vRC formation, which further enhances the preferential recruitment of RNA Pol II to viral promoters, enabling efficient viral gene transcription. We propose that this S phase arrest and the hijacking of RNA Pol II represent a novel mechanism by which AnHV-1 enhances viral transcription, offering a unique survival strategy compared to the known strategy in herpesviruses. These findings expand our understanding of herpesvirus-host interactions and highlight potential targets for antiviral strategies.

Antiviral effects and mechanism of Qi pi pill against influenza viruses.

Qi pi pill (QPP), which contains Renshen, Baizhu, Fuling, Gancao, Chenpi, Shanyao, Lianzi, Shanzha, Liushenqu, Maiya, and Zexie, was recommended for preventing and treating COVID-19 in Shandong Province (China). However, the mechanism by which QPP treats infectious diseases remains unclear. This study aims to investigate the therapeutic effect of QPP invitro and on acute influenza infection in mice, exploring its mechanism of action against influenza A virus (IAV). The invitro activity of QPP was assessed using dose-response curve analysis and titer reduction assay, and its antiviral mechanism was identified invitro by real-time polymerase chain reaction (PCR), time-of-addition, and enzymatic assays. The antiviral efficacy of QPP was further evaluated invivo using BALB/c mice infected with IAV. At the same time, each single Chinese herbal medicine in QPP was evaluated to preliminarily identify those with antiviral effects. In vitro results showed that QPP exhibited a higher potency antiviral effect against both influenza A and B viruses, inhibiting viral RNA replication and release by targeting RNA-dependent RNA polymerase and neuraminidase. Additionally, QPP significantly decreased the expression of inflammatory cytokines in A549 cells. Invivo study revealed that QPP significantly reduced the lung index and viral load in lung tissue of mice infected with IAV. Renshen, Gancao, Zexie, and Lianzi were the Chinese herbal medicines from QPP that showed anti-IAV activity. The antiviral activity of QPP targets IAV replication and release, cytokine modulation in host cells, and provides protection in mice with acute influenza infection.

Efficacy and safety of SHEN26, a novel oral small molecular RdRp inhibitor for COVID-19 treatment: a multicenter, randomized, double-blinded, placebo-controlled, phase II clinical trial

BackgroundSHEN26 (ATV014) is an oral RNA-dependent RNA polymerase (RdRp) inhibitor with potential anti-SARS-CoV-2 activity. Safety, tolerability, and pharmacokinetic characteristics were verified in a Phase I study. This phase II study aimed to verify the efficacy and safety of SHEN26 in COVID-19 patients.MethodsThis was a multicenter randomized double-blind placebo-controlled study. Mild-to-moderate adult patients with COVID-19 were recruited and randomly assigned to the high-dose (400 mg), low-dose (200 mg), or placebo groups (1:1:1). The primary outcome measure was “changes in RNA levels on Day seven (D7)”. The second outcome measures were “changes of RNA levels on D3, D5, D10, D28,” “Time of clearance of virus.”ResultsA total of 91 patients were recruited in this study between December 08, 2022, and January 27, 2023. Twelve patients dropped out due to a lack of examination results. Finally, the data of 79 patients (24 in the placebo group, 31 in the 200 mg group, and 24 in the 400 mg group) were analyzed. No significant differences in the baseline data were observed between the groups. The changes of viral load were significantly higher on D3 (P = 0.0119), and D5 (P = 0.0120) in 400 mg group (vs. placebo group), and the difference value achieved 1.06 log10 copies/mL on D3 and 1.21 log10 copies/mL on D5. No significant difference was found in the viral clearance time between SHEN26 administrating groups and placebo groups. Administration of SHEN26 did not enhance drug-related ADEs and did not induce ADEs, and ADE inducing drug withdrawal, dose reduction, or death. Moreover, SHEN26 did not worsen the renal function.ConclusionsOur findings indicate a better efficacy of a high dose (400 mg) for COVID-19 treatment. These preliminary data on the efficacy and safety provide useful information and a working basis for further verification and development of SHEN26 as a novel oral small-molecule antiviral drug for treating COVID-19.

Remdesivir and Obeldesivir Retain Potent Antiviral Activity Against SARS-CoV-2 Omicron Variants

As new SARS-CoV-2 variants continue to emerge, it is important to evaluate the potency of antiviral drugs to support their continued use. Remdesivir (RDV; VEKLURY®) an approved antiviral treatment for COVID-19, and obeldesivir (ODV) are inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase Nsp12. Here we show these two compounds retain antiviral activity against the Omicron variants BA.2.86, BF.7, BQ.1, CH.1.1, EG.1.2, EG.5.1, EG.5.1.4, FL.22, HK.3, HV.1, JN.1, JN.1.7, JN.1.18, KP.2, KP.3, LB.1, XBB.1.5, XBB.1.5.72, XBB.1.16, XBB.2.3.2, XBC.1.6, and XBF when compared with reference strains. Genomic analysis identified 29 Nsp12 polymorphisms in these and previous Omicron variants. Phenotypic analysis of these polymorphisms confirmed no impact on the antiviral activity of RDV or ODV and suggests Omicron variants containing these Nsp12 polymorphisms remain susceptible to both compounds. These data support the continued use of RDV in the context of circulating SARS-CoV-2 variants and the development of ODV as an antiviral therapeutic.

Pharmacological, computational, and mechanistic insights into triptolide's role in targeting drug-resistant cancers.

As a promising candidate for tackling drug-resistant cancers, triptolide, a diterpenoid derived from the Chinese medicinal plant Tripterygium wilfordii, has been developed. This review summarizes potential antitumor activities, including the suppression of RNA polymerase II, the suppression of heat shock proteins (HSP70 and HSP90), and the blockade of NF-kB signalling. Triptolide is the first known compound to target cancer cells specifically but spare normal cells, and it has success in treating cancers that are difficult to treat, including pancreatic, breast, and lung cancers. It acts against the tolerance mechanisms, including efflux pump upregulation, epithelial-mesenchymal transition, and cancer stem cells. Triptolide modulates important cascades, including PI3K/AKT/mTOR, enhancing the efficacy of conventional therapies. Nonetheless, its clinical application is constrained by toxicity and bioavailability challenges. Emerging drug delivery systems, such as nanoparticles and micellar formulations, are being developed to address these limitations. It has strong interactions with key anticancer targets, like PARP, as determined in preclinical and computational studies consistent with its mechanism of action. Early-phase clinical trials of Minnelide, a water-soluble derivative of triptolide, are promising, but additional work is necessary to optimize dosing, delivery, and safety. This comprehensive analysis demonstrates that triptolide may constitute a repurposed precision medicine tool to overcome tolerance in cancer therapy.

An evolved, orthogonal ssDNA generator for targeted hypermutation of multiple genomic loci.

Achieving targeted hypermutation of specific genomic sequences without affecting other regions remains a key challenge in continuous evolution. To address this, we evolved a T7 RNA polymerase (RNAP) mutant that synthesizes single-stranded DNA (ssDNA) instead of RNA in vivo, while still exclusively recognizing the T7 promoter. By increasing the error rate of the T7 RNAP mutant, it generates mutated ssDNA that recombines with homologous sequences in the genome, leading to targeted genomic hypermutation. This approach, termed T7RNAP mutant-assisted continuous evolution (T7ACE), functions effectively in both typical prokaryotic and eukaryotic microorganisms (Escherichia coli and Saccharomyces cerevisiae), achieving targeted hypermutations at rates 2800- and 1200-fold higher than the genomic mutation rates, respectively. Using T7ACE, we successfully evolved an eight-fold increase in tigecycline resistance within 7 days and doubled the efficiency of a xylose utilization pathway in 10 days, demonstrating the efficiency and broad applicability of this single-component tool for continuous evolution.

Yunvjian decoction attenuates lipopolysaccharide-induced acute lung injury by inhibiting NF-κB/NLRP3 pathway and pyroptosis

IntroductionYunvjian (YNJ) decoction, a classic traditional Chinese medicine prescription for inflammatory diseases, has demonstrated good therapeutic effects in the clinical treatment of pneumonia. The aim of this study was to clarify the effective ingredients and mechanism of action of YNJ on lipopolysaccharide (LPS)-induced acute lung injury (ALI).MethodsThe effects of YNJ were evaluated in a mouse model of LPS-induced ALI and in LPS-treated MLE-12 murine lung epithelial cells and RAW264.7 macrophages in vitro. The mechanism of action of YNJ on these model systems was studied using RNA sequencing, immunohistochemical analysis, immunoblotting, immunofluorescence, ELISA, and polymerase chain reaction assays. Ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was applied to identify the absorbed components of YNJ.ResultsYNJ attenuated pulmonary damage in LPS-treated mice, as evidenced by reduced protein content in bronchoalveolar lavage fluid, decreased lung wet/dry weight ratio, and improved respiratory function. Analysis of pneumonia-related lung injury samples from patients in the Gene Expression Omnibus dataset GSE40012 indicated that NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis was a primary mechanism in ALI. YNJ reduced the phosphorylation of nuclear factor-kappa B (NF-κB) and decreased the expression levels of lung NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1, and interleukin-1β levels (IL-1β) in vivo. Administration of YNJ-containing mouse serum increased cell viability and decreased malondialdehyde and reactive oxidative species contents in LPS-stimulated MLE-12 cells. YNJ-containing serum also decreased the secretion of tumor necrosis factor-α, IL-6, and IL-1β in LPS-stimulated RAW264.7 macrophages, and promoted macrophage polarization toward an M2 phenotype. A total of 23 absorbed components were identified in YNJ-containing serum. Among those, network analysis and in vitro experiments indicated that diosgenin, timosaponin BII, and mangiferin are anti-inflammatory active substances.ConclusionYNJ attenuates LPS-induced ALI in mice by inhibiting pyroptosis of lung epithelial cells and macrophages via suppression of the NF-κB/NLRP3 pathway. Our findings provide novel insights into the therapeutic effects of YNJ on ALI.

Development and characterization of a reverse genetics system for the lineage II Chicava strain of Machupo virus in a guinea pig model.

Machupo virus (MACV) is a New World mammarenavirus (hereafter referred to as "arenavirus") and the etiologic agent of Bolivian hemorrhagic fever (BHF). No vaccine or antiviral therapy exists for BHF, which causes up to 35% mortality in humans. New World arenaviruses evolve separately in different locations. To date, up to eight lineages of MACV have been identified in Bolivia. While the prototype MACV Carvallo strain belongs to lineage I discovered in the Memore Province in the 1960, the MACV lineage II strains have become the dominantly-circulating lineage in the same province since 1993. We report the development of a reverse genetics system for the MACV lineage II Chicava strain, using a pRF42 plasmid encoding the L and S segment genomic RNA under the transcriptional control of a murine DNA-dependent RNA polymerase I promoter sequence. Rescue of the recombinant MACV Chicava strain (rMACV-Chicava) was accomplished by expression of the L protein and nucleoprotein genes of the MACV Carvallo strain in trans in transfected baby hamster kidney (BHK-21) cells. We characterized the multiplication kinetics of rMACV-Chicava in African green monkey kidney epithelial Vero cells, followed by determining the virulence phenotype in outbred Hartley guinea pigs. We demonstrated that the multiplication kinetics in Vero cells, virulence phenotype in guinea pigs, and neutralizing antibody titers are indistinguishable between rMACV-Chicava and the wild-type parental virus. We conclude that rMACV-Chicava provides a useful model system to investigate the emergence of MACV lineage II strains and the guinea pig model has utility for the development of candidate vaccines and therapeutic antibodies for BHF.

SPT5 regulates RNA polymerase II stability via Cullin 3-ARMC5 recognition.

The stability of RNA polymerase II (Pol II) is tightly regulated during transcriptional elongation for proper control of gene expression. Our recent studies revealed that promoter-proximal Pol II is destabilized via the ubiquitin E3 ligase cullin 3 (CUL3) upon loss of transcription elongation factor SPT5. Here, we investigate how CUL3 recognizes chromatin-bound Pol II as a substrate. Using an unbiased proteomic screening approach, we identify armadillo repeat-containing 5 (ARMC5) as a CUL3 adaptor required for VCP/p97-dependent degradation of SPT5-depleted, chromatin-bound Pol II. Genome-wide analyses indicate that ARMC5 targets promoter-proximal Pol II in a BTB domain-dependent manner. Further biochemical analysis demonstrates that interaction between ARMC5 and Pol II requires the transcriptional cyclin-dependent kinase 9 (CDK9), supporting a phospho-dependent degradation model. We propose that defective, promoter-proximal Pol II that lacks SPT5 is rapidly eliminated from chromatin in a noncanonical early termination pathway that requires CDK9-dependent interaction with the CUL3-ARMC5 ubiquitin ligase complex.

Overexpression of the RNA-binding protein NrdA affects global gene expression and secondary metabolism in Aspergillus species.

RNA-binding protein Nrd1 plays a role in RNA polymerase II transcription termination. In this study, we showed that the orthologous NrdA is important in global mRNA expression and secondary metabolism in Aspergillus species. We constructed an nrdA conditional expression strain using the Tet-On system in Aspergillus luchuenesis mut. kawachii. Downregulation of nrdA caused a severe growth defect, indicating that NrdA is essential for the proliferation of A. kawachii. Parallel RNA-sequencing and RNA immunoprecipitation-sequencing analysis identified potential NrdA-interacting transcripts, corresponding to 32% of the predicted protein-coding genes of A. kawachii. Subsequent gene ontology analysis suggested that overexpression of NrdA affects the production of secondary metabolites. To clarify this, we constructed Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus oryzae strains overexpressing NrdA in the early developmental stage. Overexpression of NrdA reduced the production of sterigmatocystin and penicillin in A. nidulans, as well as that of helvolic acid and pyripyropene A in A. fumigatus. Moreover, it increased the production of kojic acid and reduced the production of penicillin in A. oryzae. These effects were accompanied by almost consistent changes in the mRNA levels of relevant genes. Collectively, these results suggest that NrdA is the essential RNA-binding protein, which plays a significant role in global gene expression and secondary metabolism in Aspergillus species.IMPORTANCENrd1, a component of the Nrd1-Nab3-Sen1 complex, is an essential RNA-binding protein involved in transcriptional termination in yeast. However, its role in filamentous fungi has not been studied. In this study, we characterized an orthologous NrdA in the Aspergillus species, identified potential NrdA-interacting mRNA, and investigated the effect of overexpression of NrdA on mRNA expression in Aspergillus luchuensis mut. kawachii. The results indicated that NrdA controls global gene expression involved in versatile metabolic pathways, including the secondary metabolic process, at least in the early developmental stage. We demonstrated that NrdA overexpression significantly affected the production of secondary metabolites in Aspergillus nidulans, Aspergillus oryzae, and Aspergillus fumigatus. Our findings are of importance to the fungal research community because the secondary metabolism is an industrially and clinically important aspect for the Aspergillus species.

Artificial cells with all-aqueous droplet-in-droplet structures for spatially separated transcription and translation

The design of functional artificial cells involves compartmentalizing biochemical processes to mimic cellular organization. To emulate the complex chemical systems in biological cells, it is necessary to incorporate an increasing number of cellular functions into single compartments. Artificial organelles that spatially segregate reactions inside artificial cells will be beneficial in this context by rectifying biochemical pathways. Here, we develop artificial cells with all-aqueous droplet-in-droplet structures that separate transcription and translation processes like the nucleus and cytosol in eukaryotic cells. This architecture uses protein-based inner droplets and aqueous two-phase outer compartments, stabilized by colloidal emulsifiers. The inner droplet is designed to enrich DNA and RNA polymerase for transcription, coupled to translation at the outer droplet via mRNA-mediated cascade reactions. We show that these processes proceed independently within each compartment, maintaining genotype-phenotype correspondence. This approach provides a practical tool for exploring complex systems of artificial organelles within large ensembles of artificial cells.

Reaction and interaction dynamics of azobenzene-tethered DNA with T7 RNA polymerase.

Reaction and interaction dynamics of azobenzene-tethered DNA (photoresponsive DNA) with T7 RNA polymerase (T7RNAP) were studied after photoisomerization of azobenzene from the cis- to trans-forms using the transient grating (TG) and time-resolved fluorescence polarization techniques. Two types of photoresponsive DNA were examined: AzoPBD, tethered at the protein binding site, and AzoTATA, tethered at the unwinding site. A diffusion change was observed after photoexcitation of cis-AzoPBD within 1 ms, and this change is explained in terms of a structural change from a bent to an extended conformation upon the cis-to-trans photoisomerization. The association and dissociation rates of cis- and trans-AzoPBD-T7RNAP complex were measured by the fluorescence polarization measurements, and the dissociation constants for cis- and trans-AzoPBD were respectively determined to be 3.9 μM and 21 μM. The result indicates that trans-AzoPBD is dissociated upon photoexcitation of cis-AzoPBD-T7RNAP complex. The efficient dissociation is mainly caused by a small association rate constant. The time-resolved diffusion measurement showed a conformational change with a time constant of 2.4 ms and a dissociation reaction with a slower rate, which depends on the concentration of T7RNAP. Although AzoTATA does not exhibit significant structural changes upon isomerization, a diffusion change was observed upon photoexcitation of AzoTATA-T7RNAP. The origin is attributed to changes from the unwound to closed states of AzoTATA, but AzoTATA does not dissociate from T7RNAP. These findings highlight a critical role of the azobenzene insertion position in modulating DNA-T7RNAP interaction dynamics, providing new insights into light-regulated transcription control.

Influenza A virus NS2 protein acts on vRNA-resident polymerase to drive the transcription to replication switch.

The heterotrimeric RNA-dependent RNA polymerase (RdRp) of influenza A virus catalyzes viral RNA transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA) by adopting different conformations. A switch from transcription to replication occurs at a relatively late stage of infection. We recently reported that the viral NS2 protein, expressed at later stages from a spliced transcript of the NS segment messenger RNA (mRNA), inhibits transcription, promotes replication and plays a key role in the transcription-to-replication switch. In this study, we performed comprehensive functional analyses to elucidate how NS2 promotes viral genome replication. Using a cell-based single-step RNP reconstitution assay, we found that NS2 specifically promotes the first-step vRNA-to-cRNA synthesis. Further investigation revealed that this promotion is tightly associated with the intrinsic properties of the 3'-vRNA promoter. Employing a highly sensitive complementation reporter assay, we demonstrated that NS2 associates more strongly with the vRNA-resident RdRp than the cRNA-resident RdRp. These findings were further validated through in vitro replication analyses. We, therefore, propose that influenza A virus NS2 protein targets vRNA-resident RdRp to drive the transcription-to-replication switch during infection.

KorB switching from DNA-sliding clamp to repressor mediates long-range gene silencing in a multi-drug resistance plasmid.

Examples of long-range gene regulation in bacteria are rare and generally thought to involve DNA looping. Here, using a combination of biophysical approaches including X-ray crystallography and single-molecule analysis for the KorB-KorA system in Escherichia coli, we show that long-range gene silencing on the plasmid RK2, a source of multi-drug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We show that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters up to 1.5 kb away. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed clamp state. DNA-bound KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Together, our findings explain the mechanistic basis for KorB role switching from a DNA-sliding clamp to a co-repressor and provide an alternative mechanism for long-range regulation of gene expression in bacteria.

Melatonin Modulates ZAP70 and CD40 Transcripts via Histone Modifications in Canine Ileum Epithelial Cells

Melatonin (MLT), produced by the pineal gland and other tissues, is known for its anti-inflammatory effects, particularly in regulating inflammatory markers and cytokines in intestinal cells. Our study aimed to investigate how MLT influences the expression of inflammatory genes through histone modification in canine ileum epithelial cells (cIECs). In our experiment, cIECs were cultured and divided into a control group (CON) and an MLT-treatment group. MLT did not significantly affect cell growth or death in cIECs compared to the CON. However, MLT treatment led to an upregulation of CD40, ZAP70, and IL7R and a downregulation of LCK, RPL37, TNFRSF13B, CD4, CD40LG, BLNK, and CIITA at the mRNA expression level. Moreover, MLT significantly altered the NF-kappa B signaling pathway by upregulating genes, such as CD40, ZAP70, TICAM1, VCAMI, GADD45B, IRAK1, TRADD, RELA, RIPK1, and RELB, and downregulating PRKCB, LY96, CD40LG, ILIB, BLNK, and TNFRSF11A. Using ChIP-qPCR, we discovered that MLT treatment enhanced histone acetylation marks H3K9ac, H3K18ac, H3K27ac, and methylation marks H3K4me1 and H3K4me3 at the ZAP70 and CD40 gene loci (p < 0.05). Additionally, the enrichment of RNA polymerase II and phosphorylated Ser5 pol-II at these loci was increased in MLT-treated cells (p < 0.05), indicating heightened transcriptional activity. In conclusion, our findings suggest that MLT mitigates inflammation in cIECs by modulating the transcription of ZAP70 and CD40 through histone modifications, offering potential therapeutic insights for inflammatory bowel diseases.

Identification of Benzothiophene-Derived Inhibitors of Flaviviruses by Targeting RNA-Dependent RNA Polymerase

Flaviviruses such as Dengue, West Nile, and Zika viruses are mosquito-borne RNA viruses that can cause serious diseases in humans. To develop effective drugs for treating these viruses’ infections, we create a new approach for developing common or shared drugs that may work for several different viral species of flaviviruses. It is based on the conserved RNA-dependent RNA polymerase (RdRp), which is the key enzyme for viral replication. We built up a common structure of RdRps (POLcon) from their consensus sequence. A conserved Triple-D structural motif was identified at the active site of POLcon that has been used for virtual compound screening. We have identified three inhibitors that have potent activities against Dengue, West Nile, and Zika viruses. All these three inhibitors are Benzothiophene derivatives. This is the first report of Benzothiophene-derived compounds as inhibitors for flaviviruses. Furthermore, our approach has provided a proof-of-concept that it is feasible to identify shared drugs for several different viral species of flaviviruses.