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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Structure of the WYL-domain containing transcription activator, DriD, in complex with ssDNA effector and DNA target site

Transcription regulators play central roles in orchestrating responses to changing environmental conditions. Recently the Caulobacter crescentus transcription activator DriD, which belongs to the newly defined WYL-domain family, was shown to regulate DNA damage responses independent of the canonical SOS pathway. However, the molecular mechanisms by which DriD and other WYL-regulators sense environmental signals and recognize DNA are not well understood. We showed DriD DNA-binding is triggered by its interaction with ssDNA, which is produced during DNA damage. Here we describe the structure of the full-length C. crescentus DriD bound to both target DNA and effector ssDNA. DriD consists of an N-terminal winged-HTH (wHTH) domain, linker region, three-helix bundle, WYL-domain and C-terminal WCX-dimer domain. Strikingly, DriD binds DNA using a novel, asymmetric DNA-binding mechanism that results from different conformations adopted by the linker. Although the linker does not touch DNA, our data show that contacts it makes with the wHTH are key for specific DNA binding. The structure indicates how ssDNA-effector binding to the WYL-domain impacts wHTH DNA binding. In conclusion, we present the first structure of a WYL-activator bound to both effector and target DNA. The structure unveils a unique, asymmetric DNA binding mode that is likely conserved among WYL-activators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular Regulatory Mechanisms Drive Emergent Pathogenetic Properties of Neisseria gonorrhoeae

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, with an estimated 87 million annual cases worldwide. N. gonorrhoeae predominantly colonizes the male and female genital tract (FGT). In the FGT, N. gonorrhoeae confronts fluctuating levels of nutrients and oxidative and non-oxidative antimicrobial defenses of the immune system, as well as the resident microbiome. One mechanism utilized by N. gonorrhoeae to adapt to this dynamic FGT niche is to modulate gene expression primarily through DNA-binding transcriptional regulators. Here, we describe the major N. gonorrhoeae transcriptional regulators, genes under their control, and how these regulatory processes lead to pathogenic properties of N. gonorrhoeae during natural infection. We also discuss the current knowledge of the structure, function, and diversity of the FGT microbiome and its influence on gonococcal survival and transcriptional responses orchestrated by its DNA-binding regulators. We conclude with recent multi-omics data and modeling tools and their application to FGT microbiome dynamics. Understanding the strategies utilized by N. gonorrhoeae to regulate gene expression and their impact on the emergent characteristics of this pathogen during infection has the potential to identify new effective strategies to both treat and prevent gonorrhea.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Bacteroides expand the functional versatility of a universal transcription factor and transcribed DNA to program capsule diversity

This record contains a list of Snakemake rules (Snakefile) to process a raw *.fastq.gz (from GEO record: GSE281607) using Cutadapt, map a processed *.fastq file using Bowtie, convert mapped reads to *.bed files using Samtools and Bedtools, and count the occurance of each Bacteriodes fragilis str NCTC 9343 genome position anywhere in a mapped read and as the 3' end of a mapped read using Bedtools. It further contains *ebwt files to assist with Bowtie mapping, *genome file to be used with Bedtools, a *.fasta file (useful to regenerate *ebwt files), and the completely processed *bed and *txt read files (generated by Snakemake and R) as references. In addition, a *.pdb file is included and represents the predicted interaction between UpeY and UpaZ.

Landick, Robert↗
DOE OSTI2021

Bubble lifetimes in DNA gene promoters and their mutations affecting transcription

Relative lifetimes of inherent double stranded DNA openings with lengths up to ten base pairs are presented for different gene promoters and corresponding mutants that either increase or decrease transcriptional activity, in the framework of the Peyrard-Bishop-Dauxois model. Extensive microcanonical simulations are used, with energies corresponding to physiological temperature. The bubble lifetime profiles along the DNA sequences demonstrate a significant reduction of the average lifetime at the mutation sites when the mutated promoter decreases transcription, while a corresponding enhancement of the bubble lifetime is observed in the case of mutations leading to increased transcription. The relative difference of bubble lifetimes between the mutated and the wild type promoters at the position of mutation varies from 20% to more than 30% as the bubble length is decreasing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

DNA affinity purification sequencing and transcriptional profiling reveal new aspects of nitrogen regulation in a filamentous fungus

Significance Microorganisms have evolved transcriptional networks to prioritize utilization of available nutrient sources. For filamentous fungi, such as Neurospora crassa , this entails distinguishing between a variety of organic and inorganic nitrogen sources. Here, we transcriptionally profiled the response of N. crassa to a variety of nitrogen sources and used DNA affinity purification sequencing to characterize the role of regulatory genes and their direct downstream targets. We identified a transcription factor responsible for regulating genes involved in amino acid and mannose metabolism. By comparing the genes regulated by transcription factors that regulate specific nitrogen utilization pathways and transcription factors that regulate utilization of all nitrogen sources that require metabolic processing before utilization, we revealed aspects of the nitrogen regulatory network.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP 6 ) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP 6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored to this core by the N-terminus of Sth1. Our cryo-EM analysis of RSC bound to a nucleosome core particle shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of the Rsc8 SWIRM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors

Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo- versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4 cis -elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure and flexibility of the yeast NuA4 histone acetyltransferase complex

The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the histone acetyltransferase (HAT) and Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1, and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4, and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome-binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggest a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Comparative transcriptome analysis of cells from different areas reveals ROS responsive mechanism at sclerotial initiation stage in Morchella importuna

Morels are some of the most highly prized edible and medicinal mushrooms, with great economic and scientific value. Outdoor cultivation has been achieved and expanded on a large scale in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and previous reports indicated that reactive oxygen species (ROS) play an important role. However, ROS response mechanisms at sclerotial initiation (SI) stage are poorly understood. In this study, comparative transcriptome analyses were performed with sclerotial and hyphal cells at different areas in the same plate at SI stage. Gene expression was significantly different at SI stage between sclerotial formation and mycelia growth areas. GO and KEGG analyses indicated more vigorous metabolic characteristics in the hyphae area, while transcription process, DNA repair, and protein processing were enriched in sclerotial cells. Gene expression related to H 2 O 2 production was high in the hyphae area, while expression of H 2 O 2 -scavenging genes was high in sclerotial cells, leading to a higher H 2 O 2 concentration in the hyphal region than in the sclerotium. Minor differences were observed in gene expression of H 2 O 2 -induced signaling pathway in sclerotial and hyphal cells; however, expression levels of the target genes of transcription factor MSN2, important in the H 2 O 2 -induced signaling pathways, were significantly different. MSN2 enhanced stress response regulation in sclerotia by regulating these target genes. Small molecular HSPs were also found upregulated in sclerotial cells. This study indicated that sclerotial cells are more resistant to ROS stress than hyphal cells through transcriptional regulation of related genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural Basis for EPC1-Mediated Recruitment of MBTD1 into the NuA4/TIP60 Acetyltransferase Complex

MBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we present the crystal structure of the MBTD1-EPC1 complex, revealing a hydrophobic C-terminal fragment of EPC1 engaging the MBT repeats of MBTD1 in a site distinct from the H4K20me binding site. Different cellular assays validate the physiological significance of the key residues involved in the MBTD1-EPC1 interaction. Our study provides a structural framework for understanding the mechanism by which MBTD1 recruits the NuA4/TIP60 acetyltransferase complex to influence transcription and DNA repair pathway choice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Single cell heterogeneity in influenza A virus gene expression shapes the innate antiviral response to infection

Viral infection outcomes are governed by the complex and dynamic interplay between the infecting virus population and the host response. It is increasingly clear that both viral and host cell populations are highly heterogeneous, but little is known about how this heterogeneity influences infection dynamics or viral pathogenicity. To dissect the interactions between influenza A virus (IAV) and host cell heterogeneity, we examined the combined host and viral transcriptomes of thousands of individual cells, each infected with a single IAV virion. We observed complex patterns of viral gene expression and the existence of multiple distinct host transcriptional responses to infection at the single cell level. We show that human H1N1 and H3N2 strains differ significantly in patterns of both viral and host anti-viral gene transcriptional heterogeneity at the single cell level. Our analyses also reveal that semi-infectious particles that fail to express the viral NS can play a dominant role in triggering the innate anti-viral response to infection. Altogether, these data reveal how patterns of viral population heterogeneity can serve as a major determinant of antiviral gene activation.

59 BASIC BIOLOGICAL SCIENCES↗