Nucleic Acids Research最新文献

In eukaryotic translation initiation, the 48S preinitiation complex (PIC) scans the 5' untranslated region of mRNAs to search for the cognate start codon (AUG) with assistance from various eukaryotic initiation factors (eIFs). Cognate start codon recognition is precise, rejecting near-cognate codons with a single base difference. However, the structural basis of discrimination of near-cognate start codons was not known. We have captured multiple yeast 48S PICs with a near-cognate AUC codon at the P-site, revealing that the AUC codon induces instability in the codon-anticodon at the P-site, leading to a disordered N-terminal tail of eIF1A. Following eIF1 dissociation, the N-terminal domain of eIF5 fails to occupy the vacant eIF1 position, and eIF2β becomes flexible. Consequently, 48S with an AUC codon is less favourable for initiation. Furthermore, we observe hitherto unreported metastable states of the eIF2-GTP-Met-tRNAMet ternary complex, where the eIF2β helix-turn-helix domain may facilitate eIF5 association by preventing eIF1 rebinding to 48S PIC. Finally, a swivelled head conformation of 48S PIC appears crucial for discriminating incorrect and selection of the correct codon-anticodon pair during translation initiation.

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanism for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrate that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we show that targeting dCas9 to telomeres using sgTelo impedes DNA replication at telomeres and induces a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigate the genome-wide features of telomeres in the dCas9/sgTelo cells and observe a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-. Consistently, we also observe an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncover many interesting molecular and structural features of the ITCB and demonstrate that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.

Bacterial and archaeal genomes encompass numerous operons that typically consist of two to five genes. On larger scales, however, gene order is poorly conserved through the evolution of prokaryotes. Nevertheless, non-random localization of different classes of genes on prokaryotic chromosomes could reflect important functional and evolutionary constraints. We explored the patterns of genomic localization of evolutionarily conserved (ancient) and variable (young) genes across the diversity of bacteria and archaea. Nearly all bacterial and archaeal chromosomes were found to encompass large segments of 100-300 kb that were significantly enriched in either ancient or young genes. Similar clustering of genes with lethal knockout phenotype (essential genes) was observed as well. Mathematical modeling of genome evolution suggests that this long-range gene clustering in prokaryotic chromosomes reflects perpetual genome rearrangement driven by a combination of selective and neutral processes rather than evolutionary conservation.

Synthetic biology enables the reprogramming of cellular functions for various applications. However, challenges in scalability and predictability persist due to context-dependent performance and complex circuit-host interactions. This study introduces an iModulon-based engineering approach, utilizing machine learning-defined co-regulated gene groups (iModulons) as design parts containing essential genes for specific functions. This approach identifies the necessary components for genetic circuits across different contexts, enhancing genome engineering by improving target selection and predicting module behavior. We demonstrate several distinct uses of iModulons: (i) discovery of unknown iModulons to increase protein productivity, heat tolerance and fructose utilization; (ii) an iModulon boosting approach, which amplifies the activity of specific iModulons, improved cell growth under osmotic stress with minimal host regulation disruption; (iii) an iModulon rebalancing strategy, which adjusts the activity levels of iModulons to balance cellular functions, significantly increased oxidative stress tolerance while minimizing trade-offs and (iv) iModulon-based gene annotation enabled natural competence activation by predictably rewiring iModulons. Comparative experiments with traditional methods showed our approach offers advantages in efficiency and predictability of strain engineering. This study demonstrates the potential of iModulon-based strategies to systematically and predictably reprogram cellular functions, offering refined and adaptable control over complex regulatory networks.

Microorganisms can produce a vast array of bioactive secondary metabolites, including DNA-intercalating agents like actinomycin D, doxorubicin, which hold great potential for cancer chemotherapy. However, discovering novel DNA-intercalating compounds remains challenging due to the limited sensitivity and specificity of conventional activity assays, which require large-scale fermentation and purification. Here, we introduced the single-molecule stretching assay (SMSA) directly to microbial cultures or extracts for discovering DNA-intercalating agents, even in trace amounts of microbial cultures (5 μl). We showed that the unique changes of dsDNA in contour length and overstretching transition enable the specific detection of intercalators from complex samples without the need for extensive purification. Applying force to dsDNA also enhanced the sensitivity by increasing both the binding affinity Ka and the quantity of ligands intercalation, thus allowing the detection of weak intercalators, which are often overlooked using traditional methods. We demonstrated the effectiveness of SMSA, identified two DNA intercalator-producing strains: Streptomyces tanashiensis and Talaromyces funiculosus, and isolated three DNA intercalators: medermycin, kalafungin and ligustrone B. Interestingly, both medermycin and kalafungin, classified as weak DNA intercalators (Ka ∼103 M-1), exhibited potent anti-cancer activity against HCT-116 cancer cells, with IC50 values of 52 ± 6 and 70 ± 7 nM, respectively.