An analytically tractable framework for transcription-extrusion coupling in chromatin loop formation.

RFA1 Inhibits Rifampicin-resistant RNA Polymerase by a Similar Mechanism as Rifampicin.

A pre-LECA origin of giant viruses as revealed by polymerase-based time tree.

mRNA vaccines and therapeutics are produced with an in vitro transcription reaction (IVT), a condensation of nucleoside triphosphates (NTPs) into a nascent mRNA chain templated by DNA and catalyzed by RNA polymerase. While tools exist to monitor NTP consumption, the role of the essential cofactor Mg2+ in IVT has not been extensively studied due to the lack of suitable analytical methods. In this study, we report the development of a high-throughput analytical method for monitoring free Mg2+ using selective fluorescent indicators. We combine this free Mg2+ monitoring method with previously reported rapid at-line monitoring of NTP and mRNA concentrations and apply it in batch and fed-batch IVT systems, varying Mg:NTP ratios and NTP concentrations to assess their effects on mRNA yield via free Mg2+. We experimentally demonstrate that free Mg2+ is critical for efficient mRNA production, with low free Mg2+ correlating with slow reaction progression or reaction stalling. For the first time, we experimentally show that under certain IVT conditions, the released free Mg2+ can be effectively utilized to continue the IVT reaction by feeding NTPs only. We also show that pyrophosphatase concentration significantly affects free Mg2+ levels and relate this to IVT kinetics and dsRNA content. Additionally, we demonstrate for the first time that free Mg2+ affects downstream DNase digestion of the pDNA template, resulting in residual pDNA when free Mg2+ levels are insufficient for DNase activity. Our findings provide an accessible methodological platform to improve the efficiency of mRNA production by optimizing IVT conditions and ensuring DNase activity.

In the context of de novo structure based drug design, the rise of in silico tools shows a promising strategy to search new molecules to address several diseases. In this work, a program with a Grammatical Evolution approach, lead to the design of 4 new molecules that can inhibit the SARS-CoV-2 RNA dependent RNA polymerase. Those were build by an exploration of a chemical space, defined previously by a pharmacophore model, with several bioisosteric fragments arrange by the grammatical evolution code into 1D molecular strings, which were then converted into 3D molecules and evaluated against the desire target using automated docking calculations. The novel drug candidates demonstrate the capacity to manifest ligand efficiencies at the biological target that are analogous to those exhibited by Remdesivir. The molecules in question also demonstrate pharmacokinetic profiles that are analogous. Two of these molecules have been observed to maintain stable interaction properties with the RNA-dependent RNA polymerase over the course of 200 nanoseconds of molecular dynamics simulation. These molecules primarily interact with the active site through weak hydrogen bonds. The software we developed for this contribution is available in the following URL: https://github.com/masotelof/GEMolecularDesign.

Fungal ribosomally synthesized and post-translationally modified peptides (F-RiPPs): Biosynthesis, genome mining, structural diversity, and translational potential as targeted anticancer ADC payloads.

Amatoxin-derived payloads and their antibody-drug conjugate: unique bicyclopeptide ADCs exhibiting targeted antitumor activity.

AlphaFold-driven structure-guided identification of NS5 RdRp-targeting antiviral leads against Kyasanur Forest Disease Virus.

Detection of lumpy skin disease virus belonging to cluster 2.5 in flies collected during the outbreak in the Republic of Korea.

Histone H3 lysine 4 trimethylation (H3K4me3) is an established hallmark of active promoters, yet the temporal hierarchy between its deposition and transcriptional initiation remains incompletely understood. Here, we employ temporal dynamic analysis of inducible human inflammatory genes to demonstrate that H3K4me3 accumulation lags significantly behind transcriptional onset. At the TNF-α and IL-1β loci, H3K4me3 enrichment succeeds the rapid recruitment of RNA polymerase II, NF-κB, and p300, appearing kinetically decoupled from the initial establishment of histone acetylation. We find that H3K4me3 deposition is attenuated by transcriptional inhibition, characterizing it as a downstream event rather than a prerequisite for initial activation. Furthermore, MLL1-mediated reduction of H3K4me3 does not impair transcriptional induction, demonstrating that this mark is dispensable for the primary onset of expression at these loci. At the constitutively active MYC locus, H3K4me3 persists after transcriptional inhibition, indicating temporal uncoupling between transcription and H3K4me3 maintenance. Our findings provide a detailed kinetic characterization of H3K4me3 loading and suggest that at these inducible genes, this modification serves as a consequence of, rather than a trigger for, transcriptional initiation.

The nucleotide addition cycle (NAC) of multisubunit DNA-dependent RNA polymerases (RNAPs) involves coordinated conformational changes in conserved active-site structural elements, including the trigger loop (TL). The TL is open (unfolded) in most RNAP structures but can close (fold) in substrate-bound (post- or pretranslocated) states of the RNAP, promoting catalysis. TL closure has been associated with closure of another conserved structural element, the Rim-Helices/F-loop (RH-FL), but the role of the RH-FL in the NAC is unclear. Antibiotic leads CBR9379 and AAP-SO2 inhibit the Escherichia coli and Mycobacterium tuberculosis RNAPs, respectively, by binding in a pocket formed by the bridge helix and RH-FL. The precise mechanism of action for these inhibitors is yet to be defined. We present cryoelectron microscopy structures showing that both compounds inhibit the RNAP NAC by preventing RH-FL closure, thereby allosterically destabilizing the closed TL. This work reveals a conserved mechanistic principle of RNAP catalysis across all domains of life and provides insight for antibiotic design.

ZMYND11 is the sole known reader of H3.3K36me3. It has been characterized as a transcriptional repressor that fine-tunes RNA polymerase II (Pol II) elongation through recognition of gene body-localized H3K36me3, based primarily on earlier ChIP-seq data showing predominant ZMYND11 occupancy at gene bodies. However, subsequent data suggest that ZMYND11 may also localize to promoter regions. Here, using CUT&Tag assay, we demonstrate that ZMYND11 robustly occupies promoter regions in mouse ESCs and MEFs, with its promoter enrichment positively correlating with gene expression levels and Pol II occupancy. We further identify formaldehyde crosslinking as a critical factor causing signal loss at transcription start sites in conventional ChIP-seq. Mechanistically, ZMYND11 deficiency reduces the pausing index of Pol II, H3.3, and H3K36me3, indicating impaired transcription initiation. ZMYND11 knockout in mouse ESCs induces transcriptomic changes, impairs cell proliferation, and aberrantly activates 2-cell-specific transcriptional programs via ROS accumulation. Our findings reveal a previously unappreciated role for ZMYND11 as a transcriptional initiator that stabilizes Pol II at promoters, establishing its essential function in maintaining embryonic stem cell homeostasis and advancing our understanding of its context-dependent transcriptional regulatory mechanisms.

Distributed transcription activity in DLX proteins defies conventional mapping of a transcription activation domain.

Cyclin-dependent kinase (CDK) 12 and its paralog, CDK13, phosphorylate RNA polymerase II, enabling transcriptional elongation. In solid tumors, CDK12 loss promotes progression by inducing replication-transcription conflict and fueling genomic instability. However, we have uncovered upregulation of CDK12 and CDK13 in ~5% of colorectal cancer (CRC) specimens, suggesting a role in cancer cell survival. Based on this, we postulated that CDK12/13 inhibition in CRC may represent a useful therapeutic strategy. To test this, we screened CDK12 and CDK12/13 inhibitors across multiple cancer cell lines and patient-derived organoids (PDO) from a range of solid tumors, demonstrating potent activity in CRC PDO. Using siRNA-mediated knockdown, we identified CDK13 as a potential mechanism of resistance to CDK12-specific inhibition. Mechanistically, CDK12/13 inhibition led to a decreased abundance of BRCA1 long transcripts, rendering cells susceptible to combination therapy with PARP inhibitors. To further assess the clinical utility of CDK12/13 inhibition, we focused on CRC, for which there is an urgent need for additional therapies. We tested the efficacy of CT7439, a novel CDK12/13 inhibitor and cyclin K degrader, which showed cytotoxicity in the low nanomolar range, reduced BRCA1 expression, and concomitant DNA damage. Together, our data support further clinical development of CDK12/13 inhibition in CRC.

Single-Molecule methods to investigate mechanisms of transcription by RNA polymerase of Mycobacterium tuberculosis.

RNA polymerase III (Pol III) is specialized for the high-throughput synthesis of short RNAs, a capability linked to its unique TFIIE- and TFIIF-like subcomplexes that are stably associated through different stages of transcription. To date, the role of a winged helix domain (WH2) of Rpc34 subunit in the TFIIE-like subcomplex during elongation has remained a conundrum because its density is consistently absent in cryo-EM structures of Pol III elongation complexes (ECs), suggesting its high conformational mobility. In this study, we employed single-molecule Förster resonance energy transfer (smFRET) and nano-positioning triangulation to characterize the dynamics and determine the position of the Rpc34-WH2 domain within transcription-competent but nontranslocating Pol III ECs. To achieve the required site-specific labeling, we developed a chemical biology framework that utilizes azido-carrying unnatural amino acid incorporation and a thiol-capping strategy to eliminate off-target alkyne-thiol cross-reactivity. With the acceptor at Rpc34-WH2 and the donor at a defined position on the DNA template as the reference point, our smFRET results reveal that Rpc34-WH2 dynamically transitions among three discrete states, corresponding to preferred positional sites in downstream, middle, and upstream regions across the DNA-binding cleft. One of these sites coincides with Rpc34-WH2's position in the preinitiation complex, indicating positional similarity across transcriptional states. Together with prior Pol I and Pol II studies, these findings establish Rpc34-WH2 as a mobile regulatory element that engages the Pol III EC through transient, weak interactions. Additionally, the bio-orthogonal labeling strategy presented here provides a robust, generalizable route for smFRET studies of large, multisubunit protein assemblies.

Silencing of human RNA polymerase I subunit A34 influences multiple cellular processes including rDNA transcription and cell migration.

Accurate RNA polymerase II (RNAPII) - dependent gene expression requires dynamic phosphorylation of the carboxy-terminal domain (CTD) of its largest subunit, Rpb1, whose heptapeptide repeats form a regulatory platform known as the CTD code. Transcription-associated cyclin-dependent kinases (tCDKs) and CTD phosphatases coordinate the phosphorylation - dephosphorylation cycle of RNAPII throughout transcription, coupling RNA synthesis to co-transcriptional processing and chromatin regulation. By controlling stage-specific modification of the CTD, these enzymes integrate RNAPII activity into broader regulatory networks. Disruption of the delicate kinase - phosphatase balance impairs transcriptional fidelity, RNA maturation, and genome stability, either directly through altered CTD phosphorylation or indirectly through associated pathways. Such alterations are increasingly associated with developmental disorders, neurodegeneration, and cancer. Here, we synthesize current knowledge of RNAPII phosphorylation dynamics, highlighting key mechanistic principles, links to human disease, and emerging therapeutic strategies targeting this central phosphorylation-dependent regulatory system.

Residual CSB activity explains mild UV-sensitive syndrome phenotype caused by CSB mutations.

An integrated sensing, adsorption, and recovery platform using engineered Escherichia coli for mercury remediation.