Abstract Bacteria produce the alarmone nucleotides ppGpp and pppGpp during stress to affect replication, transcription, and metabolism. ppGpp and pppGpp also attenuate translation by competitively binding translational GTPases to conserve resources during stress. Recently, pGpp was identified as a third alarmone, and important pathogens like Clostridioides difficile exclusively produce pGpp in response to stress. Despite its abundance as an alarmone, the precise role of pGpp in mediating stress responses is poorly understood. Here, we show that, while pGpp is a weaker inhibitor of protein synthesis than ppGpp and pppGpp in vitro, pGpp production in the model Gram-positive bacterium Bacillus subtilis leads to faster translation inhibition in vivo. pGpp production leads to fewer ribosomes engaged in translation and more hibernating ribosome dimers than (p)ppGpp production, suggesting that translation initiation is strongly inhibited. Additionally, pGpp production depletes cellular GTP more rapidly than (p)ppGpp production, which we show is sufficient for translation inhibition. Faster GTP depletion during pGpp production is also accompanied by more robust transcriptome remodeling. This work expands the model by which alarmones inhibit translation to include GTP depletion and demonstrates how different alarmone species exert varying effects on physiology.
Abstract CrRNA engineering has emerged as a pivotal strategy for extending CRISPR–Cas13a biosensing. However, structural modulation of the direct repeat (DR) region remains exceptionally challenging due to its intricate architecture and the high energetic barrier of the Cas13a–crRNA interface, which is conventionally viewed as a rigid and immutable scaffold. Here, we demonstrate that the DR region is instead a programmable topological element with unexpected structural plasticity. By systematically engineering the DR through sequence insertion and structural splitting, we identified multiple DR variants that retain robust catalytic activity. Crucially, this topological reconfiguration enables Cas13a activity to be precisely gated by unmodified nucleic acid blockers, a level of regulation unattainable with the wild-type crRNA. Building on this flexible modulation, we developed Dre-CRISPR, a DR-engineered platform that couples target-triggered DR restoration to a self-reinforcing autocatalytic loop. This self-amplifying system provides a 2 × 106-fold sensitivity enhancement over nonamplified systems. Furthermore, the Dre-CRISPR platform extends the diagnostic scope of Cas13a to a broader spectrum of analytes, ranging from microRNAs to enzymatic activities and heavy metal ions. Our findings redefine the crRNA scaffold as a versatile signaling node and provide a generalizable framework for developing high-sensitivity, self-amplifying CRISPR biosensors through topology-driven guide RNA engineering.
Protein kinases govern cellular signalling and disease progression and represent major therapeutic targets, yet comprehensive characterization across the human kinome remains hindered by data fragmentation across specialized resources. Here, we present KinaseDB (https://kinasedb.raylab.iiitd.edu.in), a freely accessible web resource integrating curated multi-omics data for 559 human protein kinases from 11 core resources, supplemented by specialized functional-site annotation datasets. KinaseDB provides structural annotations derived from experimental structures, AlphaFold models, and homology modelling; kinome-wide druggability assessments using fpocket; a provenance-aware kinase-substrate dataset comprising site-resolved and relationship-only interactions; cellular-resolution snRNA-seq expression profiles across 625 tissue-cell-type combinations; disease associations with drug-tractability annotations; and population-scale genetic variation data within a single platform. To enable target prioritization, KinaseDB introduces a kinase prioritization score (KPS) that integrates seven complementary evidence components across 69 disease contexts, comprising 38 571 kinase-disease pairs. The KPS discriminated FDA-approved kinase-inhibitor targets from IDG dark kinases with an AUROC of 0.773 (95% bootstrap CI: 0.615-0.908) and an AUPRC of 0.875 (95% bootstrap CI: 0.747-0.968). Rankings were broadly robust to the tested weight perturbations (Spearman $\rho = 0.84$-0.98 across five alternative weighting schemes). All data, bulk downloads, and a documented REST API are publicly available through KinaseDB.
Abstract Homologous recombination is a DNA repair process that requires binding of RAD51 to ssDNA at the break site. This facilitates the search for a homologous repair template on the sister chromatid, or on the homologous chromosome. How broken DNA ends loaded with RAD51 filaments are brought toward their repair template in the crowded 3D genome is currently poorly understood. This is largely due to a lack of tools to visualize homology search in living human cells. Here, we show that RAD51 and MND1, two proteins operating in homology search, become visible in long, extended structures several hours after double-stranded break formation. Using GFP-MND1 we capture these elongated foci in living human cells and reveal their highly dynamic nature as they traverse the nuclear space and gradually disassemble. We show that resolution of these structures depends on RAD54L, known for its role in RAD51-driven homology search. In addition, we find that loss of cohesin inhibits their resolution, in accordance with a role for cohesin in homology search. Thus, our data suggest that these elongated foci are visible intermediates of an active DNA repair process, and that GFP-MND1 is a powerful tool to study the dynamics of homology search in living human cells.
Abstract The minute-scale lifetime of mRNA strongly influences bacterial gene expression, whereas a robust and programmable approach to directly control the mRNA stability and topology remains elusive. Here, we develop CRESEnT (Circular RNA Expression for Stable and Enhanced Translation), a programmable in vivo mRNA circularization system based on a permuted intron–exon architecture to engineer mRNA topology. CRESEnT enables facile circularization of mRNA, which led to a substantial increase in protein expression across diverse promoters, RBS variants, genetic cargos, and bacterial hosts. Furthermore, application of CRESEnT to biosynthetic pathways increased the production of several value-added metabolites, demonstrating that mRNA circularization can be harnessed to improve the metabolic performance of microbial cell factories. Together, these results establish RNA topology engineering via circularization as a transformative axis for controlling bacterial gene expression and enhancing the functionality of microbial cells.
