Supplementary Figure S6. V0013 has a greater effect on cells activation and expansion of mouse peripheral NK compared to WT IL-15

Factor-dependent transcription activation is a key process of transcription regulation, involving complex interactions between multiple regulatory factors, RNA polymerase (RNAP), and promoter DNA, whose mechanism remains unclear. Here, we use single-molecule magnetic trapping assays and fluorescence assays to characterize the kinetics of Escherichia coli (E. coli) RNAP transcription under the regulation of a pleiotropic AraC/XylS family factor, Rob, and elucidate their underlying mechanisms. We find that Rob prebinds RNAP holoenzyme to form a binary complex to facilitate promoter search. Upon promoter recognition, Rob enhances RNAP-mediated promoter DNA unwinding to facilitate promoter escape and transition into transcription elongation. Rob activity is diversely regulated by small ligands, implying important roles of Rob C-terminal domain (CTD) on its regulatory function. Our findings shed light on the mechanisms of Rob-dependent transcription activation, which may provide insights into other transcription regulators in prokaryotes as well as eukaryotes.

Supplementary Figure S7. compared with inducing NK cell progression, V0013, weak-β IL-15 and WT IL-15 show minimal effects on human CD8+ T cells

Supplementary Figure S12. V0013 induces greater tumor inhibition and tumoral immune response than weak-β IL-15 in a PBMC transplanted moues model

The red yeast Rhodotorula toruloides is a robust lipid producer, and its biotechnology applications have been expanded through genetic engineering based on the non-homologous end joining (NHEJ) pathway. As the NHEJ pathway leads to random integration, it remains laborious in phenotypic assessment and challenging for rational strain design. To develop more efficient genetic tools for R. toruloides based on the homologous recombination (HR) pathway, we conducted a systematic evaluation of its DNA repair machinery. Overexpression of HR-related proteins, such as ScRad52, RtRad54 and ScSae2, increased the HR efficiency from less than 0.1% to 3.6%. In addition, knockout of RtKU70 and RtKU80 elevated the HR efficiency to 4.2% and 5.3%, respectively, but reduced resistance to mutagenic stress. Accordingly, we combined the two aforementioned strategies, which yielded an HR efficiency of 5.9%. Although this combined strategy failed to further elevate HR efficiency as expected, it partially alleviated the hypersensitivity to mutagenic stress caused by Ku protein inactivation. Remarkably, when the CRISPR-Cas9 system was used to introduce double-strand breaks (DSBs), HR efficiency was dramatically increased to 16%, and further overexpression of ScRAD52 led to higher HR efficiency of 75-95% across multiple genomic loci. These results will facilitate advanced genetic engineering of R. toruloides for producing value-added compounds and provide a valuable reference for the genetic manipulation of other oleaginous basidiomycetous yeasts.

Interleukin-15 (IL15) potentiates NK and T cell immunity and has huge potential for tumor immunotherapy. However, the clinical prospects of IL15 has been hindered by poor pharmacokinetics and systemic immune-related toxicities. Here, we report the development of a novel CD122-biased IL15 mutein, V0013, generated by the truncating four C-terminal amino acids (I111, N112, T113, and S114) of IL15. V0013 retains the overall cytokine structure and has intact binding to the critical CD122 receptor subunit but exhibits significantly reduced overall potency. In vivo, V0013 demonstrates a prolonged half-life, improved safety, and sustained pharmacodynamic effects. As a single agent, V0013 effectively promotes NK cell-driven antitumor immunity and enhances therapeutic efficacy of antibodies. Further mechanistic studies reveal that the interaction with specific receptor subunits dictates the function and potency of IL15 mutein. CD122 is essential for IL15-mediated signaling and prescribes its preference to NK and memory CD8+ T cells. Compared with an IL15 mutein with similar attenuation but markedly weaker CD122 binding (weak-β IL15), V0013 more closely resembles WT IL15 functions, driving superior NK cell proliferation and survival. In a tumor model engrafted with T cells, V0013 selectively enhances memory but not naïve CD8+ T cells in tumor, leading to enhanced tumor inhibition compared with weak-β IL15. RNA sequencing analysis further elucidates the critical role of CD122 in IL15 signaling and demonstrates that the mutein retaining intact CD122 binding elicits strong antitumor immunity. These findings support the therapeutic potential of the attenuated CD122-biased IL15 mutein therapy for cancer and other diseases.

Productive reiterative initiation at promoters is an alternative transcription mechanism characterized by RNA slippage relative to the template DNA and RNA polymerase, resulting in the incorporation of extra nucleotides into transcripts. A comprehensive understanding of the mechanisms underlying productive reiterative initiation, and its functional implications has been hindered due to the complexity of heterogeneous slippage transcripts. Here, we develop and employ 5' Terminal Native Elongating Transcript Sequencing (5'TNET-seq) to identify and quantify productive reiterative initiation events in Escherichia coli. Using this method, we reveal that more than half of promoters exhibit productive reiterative initiation. The conserved promoter -10 region, an appropriate spacer between the -10 region and the TSS, and the transcription initiation region associated with weak RNA-DNA hybrid stability, particularly "AAA" and "TTT" trinucleotide tracts, contribute to high productive reiterative initiation. In addition, up to four consecutive nucleotides can be added in a single cycle of productive reiterative initiation. A smaller transcription bubble is observed during productive reiterative initiation, which may stabilize the transcription initiation complex to stimulate gene transcription. Our results suggest that productive reiterative initiation emerges as an inherent transcription process regulating biological processes independent of protein regulators.

Abstract Background Tuberous sclerosis complex is a genetic disorder caused by mutations in the TSC1 or TSC2 genes, affecting multiple systems. These genes produce proteins that regulate mTORC1 activity, essential for cell function and metabolism. While mTOR inhibitors have advanced treatment, maintaining long-term therapeutic success is still challenging. For over 20 years, significant progress has linked TSC1 or TSC2 gene mutations in stem cells to tuberous sclerosis complex symptoms. Methods A comprehensive review was conducted using databases like Web of Science, Google Scholar, PubMed, and Science Direct, with search terms such as “tuberous sclerosis complex,” “TSC1,” “TSC2,” “stem cell,” “proliferation,” and “differentiation.” Relevant literature was thoroughly analyzed and summarized to present an updated analysis of the TSC1-TSC2 complex’s role in stem cell fate determination and its implications for tuberous sclerosis complex. Results The TSC1-TSC2 complex plays a crucial role in various stem cells, such as neural, germline, nephron progenitor, intestinal, hematopoietic, and mesenchymal stem/stromal cells, primarily through the mTOR signaling pathway. Conclusions This review aims shed light on the role of the TSC1-TSC2 complex in stem cell fate, its impact on health and disease, and potential new treatments for tuberous sclerosis complex. Graphical abstract

Bacteriophage Mu, a temperate phage that infects E . coli K-12 and other enteric bacteria, precisely controls its replication cycle through hijacking host RNA polymerase (RNAP) by the middle operon regulator Mor and the late gene transcription activator C. Though a dimeric arrangement and significant conformational changes are proposed for the distinct Mor/C family activators, the underlying transcription activation mechanism remains unclear. In this study, we present two cryo-EM structures of the transcription activation complex (Mor-TAC and C-TAC) with phage Mu middle and late gene promoters, respectively. Remarkably, the Mor/C activators bind to promoter DNA as a centrosymmetric tetramer rather than as the proposed dimer, concurrently stabilizing by the N-terminal dimerization domains and C-termini. The C-terminal DNA binding domains and two anti-β-strands simultaneously interact with two adjacent DNA major grooves. The activators also engage a variety of interactions with the conserved domains (αCTD, σ70R4, and β FTH) of RNAP, providing evidences for a recruitment mechanism. In addition, single-molecule FRET assays show that C significantly enhances RPitc formation, suggesting a different multi-step activation mechanism for C. Collectively, these findings reveal the unique transcription activation mechanism of tetrameric Mor/C family activators, unraveling a novel mode of phage hijacking and bacterial transcription regulation. ### Competing Interest Statement The authors have declared no competing interest.

Plants are frequently threatened by diverse stresses that severly impact their health and constrain crop productivity worldwide. Metabolic regulation serves as an important strategy for enhancing plant stress tolerance. Tryptophan, as a precursor of various plant natural products, including auxin, melatonin, and glucosinolates, plays a crucial role in maintaining plant health. To date, substantial progress has been made in elucidating tryptophan metabolism, particularly its involvement in improving plant stress tolerance. However, a systematic discussion of the crosstalk among tryptophan metabolites in protecting plants from stresses remains absent. Here, we explore tryptophan metabolism and its associated crosstalk regulation under stress conditions. We provide an overview of the biosynthesis and biofunctions of tryptophan metabolites, with a primary focus on their crosstalk in regulating plant stress resistance. The potential applications of tryptophan metabolism in stress adaptation are also examined. This work aims to establish a fundamental framework for understanding the regulatory roles of tryptophan metabolites in plant health and their mechanisms in sustainable agriculture.

Intestinal ischemia-reperfusion (II/R) injury is a common perioperative complication that occurs during severe infections, trauma, and multiple surgical procedures. II/R not only leads to localized intestinal damage but also disrupts the intestinal mucosal barrier, inducing systemic inflammatory responses and multi-organ failure, especially acute lung injury (ALI). The mechanisms are complex, involving multiple pathological processes such as oxidative stress, systemic inflammatory response, apoptosis, autophagy, and ferroptosis. During II/R, the large amount of reactive oxygen species and inflammatory factors produced rapidly activates immune cells and destroys the alveolar barrier, leading to pulmonary edema and hypoxemia, and in severe cases, acute respiratory distress syndrome (ARDS) may develop, ultimately causing respiratory failure. Current treatments include anti-inflammatory, antioxidant and anti-apoptotic drugs, as well as surgical interventions and traditional Chinese medicine. However, these methods have high drug toxicity and limited efficacy. With the development of nanomedicine, new strategies have emerged for the treatment of II/R-ALI. Nanomedicines, owing to their excellent bioavailability and targeting capabilities, can significantly enhance therapeutic outcomes and reduce side effects. This review summarizes the major mechanisms underlying II/R-ALI and discusses recent advances in the application of nanomaterials for its treatment.

The red yeast Rhodotorula toruloides emerged as a prominent host for microbial lipid production. It has been known that oxygen supply is a limiting factor for de novo lipid biosynthesis especially under high cell-density culture conditions. Bacterial hemoglobin is an oxygen-binding protein that can actively transport oxygen from the environment to metabolic processes. The aim of the present study was to address the problem of oxygen limitation during R. toruloides culture thereby enhancing cell growth and lipid synthesis. Thus, genes encoding flavohemoglobin (SHb) from Sinorhizobium meliloti and hemoglobin (VHb) from Vitreoscilla sp. were separately integrated into the genome of R. toruloides CGMCC 2.1389 by Agrobacterium-mediated transformation method. It was found that the engineered strain 4S-8 with SHB gene integration produced 49% more lipids under shake-flask culture condition than the parent strain. Under the two-stage culture process, the engineered strain 4S-8 produced 23% and 10% more cell mass than the parent strain and the strain 4V-10 with VHB gene integration, respectively, while the lipid titer of strain 4S-8 was 42% higher than that of the parent strain. These results suggested that heterologous expression of SHb in oleaginous hosts may be applicable for improved production of lipids and cell mass, which should facilitate more efficient conversion of sugars into lipids and fatty-acid derived biofuels.

α-Terpineol is a monoterpenoid alcohol that has been widely used in the flavor, fragrance, and pharmaceutical industries because of its sensory and biological properties. However, few studies have focused on the microbial production of α-terpineol. The oleaginous yeast Rhodotorula toruloides is endowed with a natural mevalonate pathway and is a promising host in synthetic biology and biorefinery. The primary objective of this work was to engineer R. toruloides for the direct biosynthesis of α-terpineol. The improvement in monoterpenoid production was achieved through the implementation of modular engineering strategies, which included the enhancement of precursor supply, blocking of downstream pathways, and disruption of competing pathways. The results of these three methods showed varying degrees of favorable outcomes in enhancing α-terpineol production. The engineered strain 5L6HE5, with competitive pathway disruption and increased substrate supply, reached the highest product titer of 1.5 mg/L, indicating that reducing lipid accumulation is an efficient method in R. toruloides engineering for terpenoid synthesis. This study reveals the potential of R. toruloides as a host platform for the synthesis of α-terpineol as well as other monoterpenoid compounds.

The enormous LysR-type transcriptional regulators (LTTRs), which are diversely distributed amongst prokaryotes, play crucial roles in transcription regulation of genes involved in basic metabolic pathways, virulence and stress resistance. However, the precise transcription activation mechanism of these genes by LTTRs remains to be explored. Here, we determine the cryo-EM structure of a LTTR-dependent transcription activation complex comprising of Escherichia coli RNA polymerase (RNAP), an essential LTTR protein GcvA and its cognate promoter DNA. Structural analysis shows two N-terminal DNA binding domains of GcvA (GcvA_DBD) dimerize and engage the GcvA activation binding sites, presenting the -35 element for specific recognition with the conserved σ70R4. In particular, the versatile C-terminal domain of α subunit of RNAP directly interconnects with GcvA_DBD, σ70R4 and promoter DNA, providing more interfaces for stabilizing the complex. Moreover, molecular docking supports glycine as one potential inducer of GcvA, and single molecule photobleaching experiments kinetically visualize the occurrence of tetrameric GcvA-engaged transcription activation complex as suggested for the other LTTR homologs. Thus, a general model for tetrameric LTTR-dependent transcription activation is proposed. These findings will provide new structural and functional insights into transcription activation of the essential LTTRs.

Sen1 is an essential helicase for factor-dependent transcription termination in Saccharomyces cerevisiae, whose molecular-motor mechanism has not been well addressed. Here, we use single-molecule experimentation to better understand the molecular-motor determinants of its action on RNA polymerase II (Pol II) complex. We quantify Sen1 translocation activity on single-stranded DNA (ssDNA), finding elevated translocation rates, high levels of processivity and ATP affinities. Upon deleting the N- and C-terminal domains, or further deleting different parts of the prong subdomain, which is an essential element for transcription termination, Sen1 displays changes in its translocation properties, such as slightly reduced translocation processivities, enhanced translocation rates and statistically identical ATP affinities. Although these parameters fulfil the requirements for Sen1 translocating along the RNA transcript to catch up with a stalled Pol II complex, we observe significant reductions in the termination efficiencies as well as the factions of the formation of the previously described topological intermediate prior to termination, suggesting that the prong may preserve an interaction with Pol II complex during factor-dependent termination. Our results underscore a more detailed rho-like mechanism of Sen1 and a critical interaction between Sen1 and Pol II complex for factor-dependent transcription termination in eukaryotes.

Abstract Plant disease, a huge burden, can cause yield loss of up to 100% and thus reduce food security. Actually, smart diagnosing diseases with plant phenomics is crucial for recovering the most yield loss, which usually requires sufficient image information. Hence, phenomics is being pursued as an independent discipline to enable the development of high-throughput phenotyping for plant disease. However, we often face challenges in sharing large-scale image data due to incompatibilities in formats and descriptions provided by different communities, limiting multidisciplinary research exploration. To this end, we build a Plant Phenomics Analysis of Disease (PlantPAD) platform with large-scale information on disease. Our platform contains 421 314 images, 63 crops and 310 diseases. Compared to other databases, PlantPAD has extensive, well-annotated image data and in-depth disease information, and offers pre-trained deep-learning models for accurate plant disease diagnosis. PlantPAD supports various valuable applications across multiple disciplines, including intelligent disease diagnosis, disease education and efficient disease detection and control. Through three applications of PlantPAD, we show the easy-to-use and convenient functions. PlantPAD is mainly oriented towards biologists, computer scientists, plant pathologists, farm managers and pesticide scientists, which may easily explore multidisciplinary research to fight against plant diseases. PlantPAD is freely available at http://plantpad.samlab.cn.

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.

Productive reiterative initiation, an alternative transcription process accompanying RNA slippage relative to the template DNA and RNA polymerase, results in the incorporation of additional nucleotides into transcripts. However, a comprehensive understanding of the mechanisms underlying productive reiterative initiation and its functional implications has been hindered due to the complexity of heterogenous slippage transcripts. Here, we develop and employ 5′ Terminal Native Elongating Transcript Sequencing (5′TNET-seq) to identify and quantify productive reiterative initiation events in Escherichia coli. 5′TNET-seq reveals that over 51% of promoters exhibit productive reiterative initiation. The conserved promoter −10 region, an appropriate spacer between the −10 region and the TSS, and the transcription initiation region associated with weak RNA-DNA hybrid stability, particularly »AAA» and »TTT» trinucleotide tracts, contribute to high productive reiterative initiation. In addition, up to four nucleotides can be added in a single cycle of productive reiterative initiation. A smaller transcription bubble is observed during productive reiterative initiation, which may stabilize the transcription initiation complex to regulate gene transcription. Our results suggest that productive reiterative initiation emerges as an inherent transcription process regulating biological processes, including cell wall synthesis, independent of protein regulators. ### Competing Interest Statement The authors have declared no competing interest.