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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

A Landing Pad System for Multicopy Gene Integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

Fatma, Zia↗
DOE OSTI2017

Systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change

Background: Long non-coding RNA (lncRNA) is a class of non-coding RNA with important regulatory roles in biological process of organisms. The systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change are still poorly understood. Here we identified 17,610 lncRNAs and calculated their expression levels based on RNA-seq of 80 individuals of Miscanthus lutarioriparius from two environments, the nearly native habitats and transplanted field, respectively. Results: LncRNAs had significantly higher expression diversity and lower expression frequency in population than protein coding mRNAs in both environments, which suggested that lncRNAs may experience more relaxed selection or divergent evolution in population compared with protein coding RNAs. In addition, the increase of expression diversity for lncRNAs was always significantly higher and the magnitude of fold change of expression in new stress environment was significantly larger than protein-coding mRNAs. These results suggested that lncRNAs may be more sensitive to environmental change than protein-coding mRNAs. Analysis of environment-robust and environment-specific lncRNA-mRNA co-expression network between two environments revealed the characterization of lncRNAs in response to environmental change. Furthermore, candidate lncRNAs contributing to water use efficiency (WUE) identified based on the WUE-lncRNA-mRNA co-expression network suggested the roles of lncRNAs in response to environmental change. Conclusion: Our study provided a comprehensive understanding of expression characterization of lncRNAs in population for M. lutarioriparius under field condition, which would be useful to explore the roles of lncRNAs and could accelerate the process of adaptation in new environment for many plants.

Expression diversity↗
DOE OSTI2024

Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection

Abstract Background Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. Methods We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. Results We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations inTP53,PLXNB3, Zinc finger genes, and fewer mutations inSDC2,CDH1,PIK3CA,SVIL, andPTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA–protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, includingKIAA1522,DCK,FOXO3,MYO9B,ARID1A,EPRS,ZC3HAV1, andRBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. Conclusions This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Oncology↗
DOE OSTI2025

Identification of shared viral sequences in peat moss metagenomes reveals elements of a possible Sphagnum core virome

Viruses are an understudied component of plant microbiomes. Identifying viruses that are shared between individual plants, or members of the “core virome”, could reveal stable viral populations with the potential to modulate the composition and function of the microbiome. Here, we examined the virome associated with Sphagnum mosses, a keystone species that has direct influence over the fate of peatland carbon stores. We analyzed bulk metagenomes and metatranscriptomes generated from Sphagnum field samples collected over a ten-month period to identify virus-like sequences shared among plants. Individual Sphagnum samples harbored distinct DNA and RNA viromes where only a small percentage (< 1%) of the total number of identified viral contigs were shared among all samples. Based on taxonomic classification, the shared viral contigs represent bacterial viruses, or phage (Caudoviricetes), as well as viruses of eukaryotes, namely nucleocytoplasmic large DNA viruses (Nucleocytoviricota) and RNA viruses (Riboviria). We linked the shared phage-like contigs to viral regions within sequenced genomes of bacterial taxa that are members of the Sphagnum core microbiome, suggesting that these contigs represent temperate phage or degraded prophage. The putative nucleocytoplasmic large DNA viruses and RNA viruses were phylogenetically diverse and showed sequence similarity to viruses associated with a broad range of hosts and environmental sources. The identification of shared viral contigs suggested that, despite the compositional heterogeneity between samples, Sphagnum mosses may harbor a core virome. Future work validating the presence of the core virome is warranted as it may aid in understanding how persistent viruses impact microbiome ecology and symbiont evolution within this climatically relevant keystone species.

Metagenomics↗
DOE OSTI2019

Structures of MERS1, the 5' processing enzyme of mitochondrial mRNAs in Trypanosoma brucei

Most mitochondrial mRNAs are transcribed as polycistronic precursors that are cleaved by endonucleases to produce mature mRNA transcripts. However, recent studies have shown that mitochondrial transcripts in the kinetoplastid protozoan, Trypanosoma brucei, are transcribed individually. Also unlike most mitochondrial mRNAs, the 5' end of these transcripts harbor a triphosphate that is hydrolyzed. This modification is carried out by a putative Nudix hydrolase called MERS1. The Nudix motif in MERS1 is degenerate, lacking a conserved glutamic acid, thus it is unclear how it may bind its substrates and whether it contains a Nudix fold. To obtain insight into this unusual hydrolase, we determined structures of apo, GTP-bound and RNA-bound T. brucei MERS1 to 2.30 Å, 2.45 Å, and 2.60 Å, respectively. The MERS1 structure has a unique fold that indeed contains a Nudix motif. The nucleotide bound structures combined with binding studies reveal that MERS1 shows preference for RNA sequences with a central guanine repeat which it binds in a single-stranded conformation. The apo MERS1 structure indicates that a significant portion of its nucleotide binding site folds upon substrate binding. Finally, a potential interaction region for a binding partner, MERS2, that activates MERS1 was identified. The MERS2-like peptide inserts a glutamate near the missing Nudix acidic residue in the RNA binding pocket, suggesting how the enzyme may be activated. Thus, the combined studies reveal insight into the structure and enzyme properties of MERS1 and its substrate-binding activities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Inferring Viral Dynamics in Chronically HCV Infected Patients from the Spatial Distribution of Infected Hepatocytes

Chronic liver infection by hepatitis C virus (HCV) is a major public health concern. Despite partly successful treatment options, several aspects of intrahepatic HCV infection dynamics are still poorly understood, including the preferred mode of viral propagation, as well as the proportion of infected hepatocytes. Answers to these questions have important implications for the development of therapeutic interventions. In this study, we present methods to analyze the spatial distribution of infected hepatocytes obtained by single cell laser capture microdissection from liver biopsy samples of patients chronically infected with HCV. By characterizing the internal structure of clusters of infected cells, we are able to evaluate hypotheses about intrahepatic infection dynamics. We found that individual clusters on biopsy samples range in size from 4-50 infected cells. In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension. These observations support the idea that HCV infection in the liver is seeded randomly (e.g. from the blood) and then spreads locally. Assuming that the amount of intracellular HCV RNA is a proxy for how long a cell has been infected, we estimate based on models of intracellular HCV RNA replication and accumulation that cells in clusters have been infected on average for less than a week. Further, we do not find a relationship between the cluster size and the estimated cluster expansion time. Lastly, our method represents a novel approach to make inferences about infection dynamics in solid tissues from static spatial data.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Transposition of HOPPLA in siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility in Brachypodium distachyon

Long terminal repeat retrotransposons (LTR-RTs) are powerful mutagens regarded as a major source of genetic novelty and important drivers of evolution. Yet, the uncontrolled and potentially selfish proliferation of LTR-RTs can lead to deleterious mutations and genome instability, with large fitness costs for their host. While population genomics data suggest that an ongoing LTR-RT mobility is common in many species, the understanding of their dual role in evolution is limited. Here, we harness the genetic diversity of 320 sequenced natural accessions of the Mediterranean grass Brachypodium distachyon to characterize how genetic and environmental factors influence plant LTR-RT dynamics in the wild. When combining a coverage-based approach to estimate global LTR-RT copy number variations with mobilome-sequencing of nine accessions exposed to eight different stresses, we find little evidence for a major role of environmental factors in LTR-RT accumulations in B. distachyon natural accessions. Instead, we show that loss of RNA polymerase IV (Pol IV), which mediates RNA-directed DNA methylation in plants, results in high transcriptional and transpositional activities of RLC_BdisC024 (HOPPLA) LTR-RT family elements, and that these effects are not stress-specific. This work supports findings indicating an ongoing mobility in B. distachyon and reveals that host RNA-directed DNA methylation rather than environmental factors controls their mobility in this wild grass model.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Transcription Start Site Associated RNAs (TSSaRNAs) Are Ubiquitous in All Domains of Life

A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (~7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX

Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Nanometer-scale siRNA carriers incorporating peptidomimetic oligomers: physical characterization and biological activity

Synthetic short interfering RNA (siRNA) oligonucleotides can trigger the RNA interference pathway and lead to selective gene silencing. Despite considerable enthusiasm and investment, formidable challenges remain that may deter translating this breakthrough discovery into clinical applications. In particular, the development of efficient, nontoxic, nonimmunogenic methods for delivering siRNA in vivo has proven to be exceptionally challenging. Thorough analysis of the relationship between the structure and function of siRNA carrier systems, both in isolation and in complex with RNA, will facilitate the design of efficient nonviral siRNA delivery vehicles. In this study, we explore the relationship between the physicochemical characteristics and the biological activity of “lipitoid” compounds as potent siRNA delivery vehicles. Lipitoids are cationic peptidomimetic oligomers incorporating a peptoid and a phospholipid moiety. Lipitoids can associate with siRNA oligonucleotides and self-assemble into spherical lipitoid-based nanoparticles (LNPs), with dimensions that are dependent upon the medium and the stoichiometric ratio between the cationic monomers of the lipitoid and anionic siRNA oligonucleotides. The morphology, gene silencing efficiency, and cytotoxicity of the siRNA-loaded LNPs are similarly sensitive to the stoichiometry of the complexes. The medium in which the LNPs are formed affects the assembled cargo particles’ characteristics such as particle size, transfection efficiency, and stability. Formation of the LNPs in the biological, serum-free medium OptiMEM resulted in LNPs an order of magnitude larger than LNPs formed in water, and were twice as efficient in siRNA transfection compared to LNPs formed in water. Inhibitor studies were conducted to elucidate the efficiency of lysosomal escape and the uptake mechanism of the siRNA-loaded LNPs. Our results suggest that these lipitoid-based, siRNA-loaded spherical LNPs are internalized through a lipid raft-dependent and dynaminmediated pathway, circumventing endosomal and lysosomal encapsulation. The lipitoid-siRNA nanospheres proved to be suitable platforms for investigating the critical parameters determining the efficiency of transfection agents, revealing the necessity for conducting characterization studies in biological media. The investigation of the LNP internalization pathway points to an alternative uptake route that bypasses the lysosome, explaining the surprisingly high efficiency of LNPs and suggesting that the uptake mechanism should be probed rather than assumed for the next generation of rationally designed transfection agents.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Genomics-enabled sensor platform for rapid detection of viruses related to disease outbreak.

Bioweapons and emerging infectious diseases pose growing threats to our national security. Both natural disease outbreak and outbreaks due to a bioterrorist attack are a challenge to detect, taking days after the outbreak to identify since most outbreaks are only recognized through reportable diseases by health departments and reports of unusual diseases by clinicians. In recent decades, arthropod-borne viruses (arboviruses) have emerged as some of the most significant threats to human health. They emerge, often unexpectedly, from cryptic transmission foci causing localized outbreaks that can rapidly spread to multiple continents due to increased human travel and trade. Currently, diagnosis of acute infections requires amplification of viral nucleic acids, which can be costly, highly specific, technically challenging and time consuming. No diagnostic devices suitable for use at the bedside or in an outbreak setting currently exist. The original goals of this project were to 1) develop two highly sensitive and specific diagnostic assays for detecting RNA from a wide range of arboviruses; one based on an electrochemical approach and the other a fluorescent based assay and 2) develop prototype microfluidic diagnostic platforms for preclinical and field testing that utilize the assays developed in goal 1. We generated and characterized suitable primers for West Nile Virus RNA detection. Both optical and electrochemical transduction technologies were developed for DNA-RNA hybridization detection and were implemented in microfluidic diagnostic sensing platforms that were developed in this project.

Brozik, Susan M↗
DOE OSTI2019

Predicting Ionizing Radiation Exposure (LDRD Final Report)

Rapid, low-cost, easy-to-use devices are needed for screening large populations potentially exposed to ionizing radiation following accidental or intentional release of radioactive materials. Measurements of radiation responsive messenger RNA (mRNA) biomarkers using fieldable devices could provide such a tool. mRNA expression level changes may be useful for discriminating between exposed individuals and the worried well and estimating the radiation dose received even days after exposure. Loop mediated isothermal amplification, or LAMP, is an established nucleic acid amplification technique that provides key advantages for use in low cost, portable instruments. We proposed to use LAMP to determine if the expression level of one or a small number of radiation responsive biomarkers can be used to discriminate unexposed cells from ionizing radiation (IR)-exposed cells. If successful, this methodology could potentially be transitioned to portable detection technology for use in population screening after large-scale events. Our approach involved designing quantitative, real-time, reverse transcriptase loop-mediated isothermal amplification (RTLAMP) assays for a panel of published IR-responsive mRNA biomarkers and control genes. This assay panel was then tested on mRNA isolated from white blood cells, lymphocyte cell lines and samples from internally dosed individuals. We used standard curves to quantify mRNA copy number along with the comparative Ct method to measure relative mRNA expression changes. We were able to generate quantitative and comparative gene expression changes for many of the samples evaluated. Although promising in its potential to measure gene expression, the technique suffered when attempting to amplify target genes with very low background RNA expression levels, in particular, FDXR in controls samples. Future work should focus on optimizing the RT-LAMP reaction for low RNA expression level targets allowing for the generation of validated assay panels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Reveal-CoV Diaagnostic Platform (Final Report)

LLNL designed and built a rapid, RT-LAMP-based molecular diagnostics platform as a potential tool to quickly diagnose COVID-19 in under one hour. This point-of-care testing approach involves an initial high temperature swab sample inactivation step followed by amplification of viral RNA using up to 5 control and pathogen-specific assays. Results are determined based on a discreet reaction color change from red to yellow but can also be determined using fluorescence detection. Testing of this prototype platform was conducted with synthetic viral RNA and dried, stabilized reagents. Buffer systems, swab selection, and assay stabilization formulations were evaluated for performance. Limits of detection were determined using RNA; however, testing was not performed with viable SARS-CoV-2 virus or clinical samples.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Generating Co-expression Networks for Three Cyanobacteria: Synechococcus sp. PCC 7942, Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803

Cyanobacteria are photosynthetic organisms capable of high growth rate and represent a promising bioplatform for harnessing the sun’s energy to make biofuel. Additionally, the process of photosynthesis absorbs CO2 from the environment. Understanding the metabolic processes involved in photosynthesis could lead to solutions to the recent rise of CO2 concentration in Earth’s atmosphere and the associated climate change. More research on the transcriptional regulation of these cells is needed to learn how to harness the untapped potential of cyanobacteria for these applications. Transcriptional analysis via RNA-seq provides an understanding of how gene expression changes at the mRNA level under diverse growing conditions. I systematically collected and analyzed RNA-Seq data obtained under a variety of conditions and available on the NCBI database for three cyanobacteria model organisms: Synechococcus elongatus sp. PCC 7942, Synechococcus sp. PCC 7002, and Synechocystis sp. PCC 6803. For each organism, the data was mapped to a reference genome to characterize the RNA expression profile. Samples were checked for quality based on the number of reads and the correlation of the expression profile between labeled replicates. All samples were transformed into transcripts per million reads, followed by a log transformation to account for the wide range of sample sizes. Gene co-expression networks were generated and analyzed for each species using Cytoscape. These networks provide a base level of gene expression for each species. The network topology and high-betweeness nodes of these networks need to be analyzed further to provide insight on potential ways to harness cyanobacteria genetics. Additionally, these datasets can be used together to form a core genome network analysis- one that includes only the genes that are homologous between the three species. This project has prepared the way for a more in-depth study on photosynthetic microbes on a genetic level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Human Liver Epithelium Response to HCoV-229E Infection Epigenomics (ACS-DP4)

The purpose of this experiment was to evaluate how wild-type Human coronavirus strain 229E (HCoV-229E) infection alters chromatin accessibility in infected cells only. Sample data was obtained for mock and infected (standard and UV-inactivated) immortalized human liver cells (HuH-7) and collected 24 hrs. post infection. Samples were processed using assay for transposase-accessible chromatin using high-throughput sequencing (ATAC-Seq) and generated bar coded library samples were evaluated for RNA sequencing (RNA-Seq) expression analysis. Processed ATAC-Seq datasets are openly accessible from the download button and contain secondary processed RNA-Seq results files and supporting metadata materials. Data download includes a sample naming key, infection titer metadata, normalized counts, and relevant computational source code information supporting data transparency and reuse.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

COVID-19 trends at the University of Tennessee: predictive insights from raw sewage SARS-CoV-2 detection and evaluation and PMMoV as an indicator for human waste

Wastewater-based epidemiology (WBE) has become a valuable tool for monitoring the prevalence of SARS-CoV-2 on university campuses. However, concerns about effectiveness of raw sewage as a COVID-19 early warning system still exist, and it’s not clear how useful normalization by simultaneous comparison of Pepper Mild Mottle Virus (PMMoV) is in addressing variations resulting from fecal discharge dilution. This study aims to contribute insights into these aspects by conducting an academic-year field trial at the student residences on the University of Tennessee, Knoxville campus, raw sewage. This was done to investigate the correlations between SARS-CoV-2 RNA load, both with and without PMMoV normalization, and various parameters, including active COVID-19 cases, self-isolations, and their combination among all student residents. Significant positive correlations between SARS-CoV-2 RNA load a week prior, during the monitoring week, and the subsequent week with active cases. Despite these correlations, normalization by PMMoV does not enhance these associations. These findings suggest the potential utility of SARS-CoV-2 RNA load as an early warning indicator and provide valuable insights into the application and limitations of WBE for COVID-19 surveillance specifically within the context of raw sewage on university campuses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Variation of Structure and Cellular Functions of Type IA Topoisomerases across the Tree of Life

Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Homologs of Phycobilisome Abundance Regulator PsoR Are Widespread across Cyanobacteria

During chromatic acclimation (CA), cyanobacteria undergo shifts in their physiology and metabolism in response to changes in their light environment. Various forms of CA, which involves the tuning of light-harvesting accessory complexes known as phycobilisomes (PBS) in response to distinct wavelengths of light, have been recognized. Recently, a negative regulator of PBS abundance, PsoR, about which little was known, was identified. We used sequence analyses and bioinformatics to predict the role of PsoR in cyanobacteria and PBS regulation and to examine its presence in a diverse range of cyanobacteria. PsoR has sequence similarities to the β-CASP family of proteins involved in DNA and RNA processing. PsoR is a putative nuclease widespread across Cyanobacteria, of which over 700 homologs have been observed. Promoter analysis suggested that psoR is co-transcribed with upstream gene tcpA. Multiple transcription factors involved in global gene regulation and stress responses were predicted to bind to the psoR-tcpA promoter. The predicted protein–protein interactions with PsoR homologs included proteins involved in DNA and RNA metabolism, as well as a phycocyanin-associated protein predicted to interact with PsoR from Fremyella diplosiphon (FdPsoR). The widespread presence of PsoR homologs in Cyanobacteria and their ties to DNA- and RNA-metabolizing proteins indicated a potentially unique role for PsoR in CA and PBS abundance regulation.

Layer, Alicia↗