Timoshenko Beam Modeling of Realistic Wind Turbine Blades Using Mechanics of Structure Genome
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Bagla, Akshat [Purdue University]↗
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Telomere-to-telomere assemblies of chromosome 10 reveal complex adaptive variation of 3-ketoacyl-CoA-synthases in Populus trichocarpa likely driven by Helitrons
The model woody plant Populus trichocarpa displays an atypical alkene-diverse wax cuticle likely driven by copy number variation (CNV) of 3-ketoacyl-CoA synthases ( KCS ), which has been difficult to confirm with short-read assemblies. Long-read sequencing enables the development of telomere-to-telomere resources to detect cryptic variation, including CNVs, which are currently missed. Integrating this information can improve genomic prediction for breeding and provide insights into the evolutionary basis of important traits. Our analysis of 78 long-read haplotypes from chromosome 10 identified more than twice as many KCS genes as previously reported, and numerous intragenic non-synonymous substitutions. Random Forest predictive models highlighted the importance of Potri.010G079500 in producing very long chain alkenes; however, its absence did not predict previously reported alkene-deficient phenotypes. Instead, alkene levels are best predicted by the combinations of KCS copies. Additionally, amino acid substitutions clustered around ligand and donor binding pockets, suggesting they contribute to differing wax cuticle composition. Finally, each KCS gene and copy was linked to a Helitron transposon. A phylogenetic analysis suggests Helitrons are the evolutionary mechanism for generating KCS tandem arrays. Long-read generated telomere-to-telomere assemblies of P. trichocarpa chromosome 10 revealed large-effect loci critical to genetic studies that are unattainable from short-reads. This new resource produced novel insights into genome structure and function, and a novel mechanism for generating tandem gene duplication. Our results highlight that, given current challenges in annotation and assembly, detailed and focused long-read sequences are key to interpreting complex genomic regions that contain tandem copy number variants.
09 BIOMASS FUELS↗
Multi-omics analysis reveals the dynamic interplay between Vero host chromatin structure and function during vaccinia virus infection
The genome folds into complex configurations and structures thought to profoundly impact its function. The intricacies of this dynamic structure-function relationship are not well understood particularly in the context of viral infection. To unravel this interplay, here we provide a comprehensive investigation of simultaneous host chromatin structural (via Hi-C and ATAC-seq) and functional changes (via RNA-seq) in response to vaccinia virus infection. Over time, infection significantly impacts global and local chromatin structure by increasing long-range intra-chromosomal interactions and B compartmentalization and by decreasing chromatin accessibility and inter-chromosomal interactions. Local accessibility changes are independent of broad-scale chromatin compartment exchange (~12% of the genome), underscoring potential independent mechanisms for global and local chromatin reorganization. While infection structurally condenses the host genome, there is nearly equal bidirectional differential gene expression. Despite global weakening of intra-TAD interactions, functional changes including downregulated immunity genes are associated with alterations in local accessibility and loop domain restructuring. Therefore, chromatin accessibility and local structure profiling provide impactful predictions for host responses and may improve development of efficacious anti-viral counter measures including the optimization of vaccine design.
59 BASIC BIOLOGICAL SCIENCES↗
Reference-free structural variant detection in microbiomes via long-read co-assembly graphs
Motivation: The study of bacterial genome dynamics is vital for understanding the mechanisms underlying microbial adaptation, growth, and their impact on host phenotype. Structural variants (SVs), genomic alterations of 50 base pairs or more, play a pivotal role in driving evolutionary processes and maintaining genomic heterogeneity within bacterial populations. While SV detection in isolate genomes is relatively straightforward, metagenomes present broader challenges due to the absence of clear reference genomes and the presence of mixed strains. In response, our proposed method rhea, forgoes reference genomes and metagenome-assembled genomes (MAGs) by encompassing all metagenomic samples in a series (time or other metric) into a single co-assembly graph. The log fold change in graph coverage between successive samples is then calculated to call SVs that are thriving or declining. Results: We show rhea to outperform existing methods for SV and horizontal gene transfer (HGT) detection in two simulated mock metagenomes, particularly as the simulated reads diverge from reference genomes and an increase in strain diversity is incorporated. We additionally demonstrate use cases for rhea on series metagenomic data of environmental and fermented food microbiomes to detect specific sequence alterations between successive time and temperature samples, suggesting host advantage. Our approach leverages previous work in assembly graph structural and coverage patterns to provide versatility in studying SVs across diverse and poorly characterized microbial communities for more comprehensive insights into microbial gene flux.
59 BASIC BIOLOGICAL SCIENCES↗
Structural basis for a highly conserved RNA-mediated enteroviral genome replication
Abstract Enteroviruses contain conserved RNA structures at the extreme 5′ end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Å resolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3′ spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3′ spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform.
Biochemistry & Molecular Biology↗
Structure of coxsackievirus cloverleaf RNA and 3C pro dimer establishes the RNA-binding mechanism of enterovirus protease 3C pro
In positive-strand RNA viruses, the genome serves as a template for both protein translation and negative-strand RNA synthesis. Enteroviruses use the cloverleaf RNA structure at the 5′ end of the genome to balance these two processes. Cloverleaf acts as a promoter for RNA synthesis and forms a complex with viral 3CD protein, the precursor to 3C pro protease, and 3D pol polymerase. The interaction between cloverleaf and 3CD is mediated by the 3C pro domain, yet how 3C pro promotes specific RNA-binding is not clear. We report the structure of coxsackievirus cloverleaf RNA-3C pro complex, wherein two 3C pro molecules interact with cloverleaf stem-loop D. 3C pro dimer mainly recognizes the shape of the dsRNA helix through symmetric interactions, suggesting that 3C pro is a previously undiscovered type of RNA binding protein. We show that 3CD protein also dimerizes on cloverleaf RNA and binds the RNA with higher affinity than 3C pro . The structure provides insight into the RNA-binding mechanism of 3C pro or 3CD with other cis-acting replication elements.
Science & Technology - Other Topics↗
The Importance of Solution Studies for the Structural Characterization of the Enterovirus 5’ Cloverleaf
Enteroviruses initiate genomic replication via a highly conserved mechanism that is controlled by an RNA platform, also known as the 5’ cloverleaf (5’CL). Here, we present a biophysical analysis of the 5’CL conformation of three enterovirus serotypes under various ionic conditions, utilizing CD spectroscopy, size-exclusion chromatography, and small-angle X-ray scattering. In general, a tendency toward a smaller monomeric hydrodynamic radius in the presence of salts was observed, but the exact structural signature of each 5’CL varied depending upon the serotype. Rhinovirus B14 (RVB14) exhibited at least two monomeric conformations and a low propensity for dimerization, while poliovirus 1 (PV1) showed a high propensity for dimerization, which was enhanced by the presence of salts. Enterovirus D70 was observed to be somewhat intermediate, with primarily a monomeric structure, but possessing some potential for dimerization. The equilibrium between the two monomeric and the dimeric conformations is also discussed. These results indicate that the 5’CL conformation may be more complex than the current literature suggests, thus underscoring the need for a combined crystal and solution approach for the accurate representation of the 5’CL conformation, and the conformation of other RNA structural elements, under native conditions.
Virology↗
CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation
RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a–RNA–DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.
59 BASIC BIOLOGICAL SCIENCES↗
Genomic analysis of Klebsiella aerogenes circulating in New Mexico
Klebsiella aerogenes is an opportunistic pathogen and a growing cause of healthcare-associated infections, characterized by multidrug resistance and the emergence of global high-risk clones. However, regional genomic surveillance data remain limited. Here, we sought to characterize the population structure, transmission dynamics and resistance mechanisms of clinical K. aerogenes in Albuquerque, New Mexico. We sequenced 177 clinical isolates collected between 2021 and 2023. We also developed a novel, species-specific PopPUNK database to facilitate rapid, high-resolution typing. The New Mexico K. aerogenes population was diverse but dominated by two global pandemic lineages, ST93 (47.5%) and ST4 (7.9%), which were significantly enriched for the virulence factors yersiniabactin and colibactin. Genomic evidence for recent local transmission was rare, with only four putative transmission pairs identified. The resistome was characterized by intrinsic and adaptive mutations. Nearly all isolates possessed gyrA mutations associated with decreased fluoroquinolone susceptibility. Mutations in the AmpC regulator AmpD and the outer membrane porin Omp36 were common, particularly within the dominant ST93 lineage. These mutations have been associated with increased AmpC-mediated carbapenem resistance. Our findings underscore the critical importance of genomic surveillance to monitor the transmission and evolution of adaptive resistance.
59 BASIC BIOLOGICAL SCIENCES↗
Mechanism-guided engineering of a minimal biological particle for genome editing
The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.
59 BASIC BIOLOGICAL SCIENCES↗
Secure biosystems design in Saccharomyces cerevisiae establishes effective biocontainment strategies and mechanisms of escape
The widespread application of recombinant DNA and synthetic biology approaches for microbial metabolic engineering pursuits has motivated the development of biocontainment strategies, targeting safe and secure deployment of genetically modified microorganisms (GMMs). However, the design rules and mechanistic drivers governing biocontainment efficacy, as well as impacts of biocontainment upon microbial fitness, remain to be comprehensively evaluated, hindering predictive design and application of these strategies. We have developed a platform for high-resolution analysis of a transactivated kill switch in laboratory and industrial strains of Saccharomyces cerevisiae to assess modes of biocontainment escape and establish design rules for development of kill switch systems in diverse microbes. A camphor-regulated, RelE toxin system was systematically deployed to assess the impacts of differential kill switch copy number and ploidy in laboratory vs industrial strains. CRISPR-mediated integration of the biocontainment system at various loci revealed rapid escape events driven, in part, by mutations to both the Cam-transactivator (cam-TA) and RelE toxin. Genetic engineering enabled recapitulation of escape phenotypes, confirming mechanisms of escape and establishing structure-function relationships in the cam-TA system. Interestingly, genomic resequencing of escape mutants also revealed a series of off-target mutations, implicating additional modes of kill switch escape. Multi-copy integration of the kill switch system mitigated these effects by orders of magnitude, without compromising the biosynthetic capacity of the microbes, but proved insufficient to establish sustained biocontainment. The resultant data define a series of key design rules for next-generation biocontainment strategies and add to a growing foundational knowledge base targeting establishment of secure biosystems designs.
59 BASIC BIOLOGICAL SCIENCES↗
Genome resources for three modern cotton lines guide future breeding efforts
Cotton ( Gossypium hirsutum L.) is the key renewable fibre crop worldwide, yet its yield and fibre quality show high variability due to genotype-specific traits and complex interactions among cultivars, management practices and environmental factors. Modern breeding practices may limit future yield gains due to a narrow founding gene pool. Precision breeding and biotechnological approaches offer potential solutions, contingent on accurate cultivar-specific data. Here we address this need by generating high-quality reference genomes for three modern cotton cultivars (‘UGA230’, ‘UA48’ and ‘CSX8308’) and updating the ‘TM-1’ cotton genetic standard reference. Despite hypothesized genetic uniformity, considerable sequence and structural variation was observed among the four genomes, which overlap with ancient and ongoing genomic introgressions from ‘Pima’ cotton, gene regulatory mechanisms and phenotypic trait divergence. Differentially expressed genes across fibre development correlate with fibre production, potentially contributing to the distinctive fibre quality traits observed in modern cotton cultivars. These genomes and comparative analyses provide a valuable foundation for future genetic endeavours to enhance global cotton yield and sustainability.
59 BASIC BIOLOGICAL SCIENCES↗
Structural insights into transcriptional regulation by the helicase RECQL5
Abstract Transcription poses a major challenge for genome stability. The RECQL5 helicase helps safeguard genome integrity and is the only member of the human RecQ helicase family that directly binds to RNA polymerase II (Pol II) and affects its progression. While RECQL5 mitigates transcription stress in cells, the molecular mechanism underlying this phenomenon is unclear. Here, we use cryo-electron microscopy to determine the structures of stalled human Pol II elongation complexes (ECs) bound to RECQL5. Our structures reveal the molecular interactions stabilizing RECQL5 binding to the Pol II EC and highlight its role as a transcriptional roadblock. Additionally, we find that, in its nucleotide-free state, RECQL5 twists the downstream DNA in the EC and, upon nucleotide binding, undergoes a conformational change that allosterically induces Pol II toward a post-translocation state. We propose that this mechanism may help restart Pol II elongation and, therefore, contribute to reducing transcription stress.
Biochemistry & Molecular Biology↗
Cryo-EM structure of AAV2 Rep68 bound to integration site AAVS1: insights into the mechanism of DNA melting
Abstract The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions necessary for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin of replication, catalyzing the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. We report the cryo-electron microscopy structures of Rep68 bound to the adeno-associated virus integration site 1 in different nucleotide-bound states. In the nucleotide-free state, Rep68 forms a heptameric complex around DNA, with three origin-binding domains (OBDs) bound to the Rep-binding element sequence, while three remaining OBDs form transient dimers with them. The AAA+ domains form an open ring without interactions between subunits and DNA. We hypothesize that the heptameric structure is crucial for loading Rep68 onto double-stranded DNA. The ATPγS complex shows that only three subunits associate with the nucleotide, leading to a conformational change that promotes the formation of both intersubunit and DNA interactions. Moreover, three phenylalanine residues in the AAA+ domain induce a steric distortion in the DNA. Our study provides insights into how an SF3 helicase assembles on DNA and provides insights into the DNA melting process.
Jaiswal, Rahul↗
Divergent Rickettsia species exhibit distinct mechanisms of actin-based motility
Many Rickettsia species undergo actin-based motility to promote cell-cell spread during infection. Rickettsial genomes often encode two motility effectors, RickA and Sca2, which in the spotted fever group I species, Rickettsia parkeri act by activating the host Arp2/3 complex and by mimicking eukaryotic formins, respectively. The function of RickA and Sca2 orthologs in the distantly related species Rickettsia bellii was unclear. We report that R. bellii RickA activates the host Arp2/3 complex but has no discernible role in bacterial motility. The R. bellii Sca2 ortholog, Sca2/6, nucleates and elongates actin with a flexible structure and an unusual actin monomer-binding motif in a mechanism distinct from formins or other microbial actin nucleators. R. bellii motility is solely correlated with Sca2/6 localization and, compared with R. parkeri motility, is slow and meandering, generating distinctly organized actin tails. The evolutionary flexibility in the mechanism and regulation of rickettsial actin-based motility suggests similar adaptability for other microbes.
Bacher, Meghan C↗
Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria
Abstract Background Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. Results Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. Conclusions Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.
59 BASIC BIOLOGICAL SCIENCES↗
Enhancing Biopreparedness through a Model System to Understand the Molecular Mechanisms that Lead to Pathogenesis and Disease Transmission: NW-BRaVE
The science of biopreparedness to counter biological threats hinges on understanding the fundamental principles and molecular mechanisms that lead to pathogenesis and disease transmission. Our vision to address this challenge is to create a powerful and user-friendly platform to elucidate the fundamental principles of how molecular interactions drive pathogen-host relationships and host shifts. We will enable groundbreaking discoveries by integrating a wide range of structural, genomics, proteomics, and other advanced omics measurements, along with evolutionary and artificial intelligence predictions. To make sure the system is applicable to real-world problems, we will develop it in the context of a tractable model system, the small, abundant, and accessible photosynthetic cyanobacteria and their constantly co-adapting viral pathogens, cyanophages. This model will maintain the system’s applicability to real-world problems and techniques, but the overall focus will be on elucidating general principles of detecting, assessing, and surveilling molecular interaction, adaptation, and coevolution that are system agnostic and therefore extensible to other viral-host interactions. Our overall objectives are to (1) identify the molecular complexes that comprise the cyanobacteria redox macromolecular subsystem and how they dynamically change with bacteriophage infection in situ, using cryo-electron tomography; (2) profile regulatory changes during infection using proteomics, multiomics, and experimental validation, and integrate the data with in situ structures; (3) use genomics and metagenomics to determine environmental and population factors across time scales that impact the interactions between marine cyanobacteria and their cyanophage parasites, predicting the evolutionary origins of in situ structural and functional interactions, convergence and coevolution; and (4) develop a data integration and transformation platform that facilitates the integration of in situ, proteomic, and evolutionary measurements of molecular interactions to surveil diverse hosts and parasites in various environmental contexts. These objectives address Focus Area 2 Reveal Molecular Interactions Across Biological Scales for Design of Targeted Interventions. Our powerful and user-friendly platform will enhance connections between the often-siloed fields of structure, molecular phenotype, and evolutionary genomics that are key to biopreparedness, but in need of integration (Figure 1). We will build an integrated navigation tool to facilitate the effective use of globally distributed experimental data for integrated analysis and predictive modeling. The project will develop, implement, and test a platform to assess host-pathogen molecular interactions, adaptation to hosts and host shifts, and coevolution between hosts and pathogens, successfully impacting the research community by revolutionizing abilities to study any host-pathogen interaction, encourage diverse community contributions, and gain fundamental insights into how proteins adapt to new contexts. This ability will be critical for designing early interventions to address future threats. We will build surveillance training capability, aiming for a fair and equitable response to future pandemics and biothreats.
59 BASIC BIOLOGICAL SCIENCES↗
Supporting Information for manuscript: “A latitudinal gradient in S/G lignin monomer ratio driven by laccase in natural poplar variants”
Lignin composition plays a crucial role in plant structural integrity and environmental adaptation. However, the genetic and molecular mechanisms underlying natural variation in lignin composition remain poorly understood. This study investigates the syringyl-to-guaiacyl (S/G) lignin monomer ratio across a natural population of Populus trichocarpa spanning a latitudinal gradient along the Northwest coast of North America. By integrating biochemical, genomic, and geographic analysis, we identify key gene variants associated with S/G ratio differences. These datasets provide valuable insights into the evolutionary and functional genomics of lignin composition and serve as a resource for developing poplar variants optimized for forestry and bioenergy applications.
Poplar, lignin composition, laccases, latitude, ad↗