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At least 1,045 records · Page 58
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Structures of the DarR transcription regulator reveal unique modes of second messenger and DNA binding

The mycobacterial repressor, DarR, a TetR family regulator (TFR), was the first transcription regulator shown to bind c-di-AMP. However, the molecular basis for this interaction and the mechanism involved in DNA binding by DarR remain unknown. Here we describe DarR-c-di-AMP and DarR-DNA structures and complementary biochemical assays. The DarR-c-di-AMP structure reveals a unique effector binding site for a TFR, located between DarR dimer subunits. Strikingly, we show this motif also binds cAMP. The location of the adenine nucleotide binding site between subunits suggests this interaction may facilitate dimerization and hence DNA binding. Indeed, biochemical assays show cAMP enhances DarR DNA binding. Finally, DarR-DNA structures reveal a distinct TFR DNA-binding mechanism involving two interacting dimers on the DNA. Thus, the combined data unveil a newly described second messenger binding motif and DNA binding mode for this important family of regulators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation

Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei ( Mm ) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Myosin-binding protein C regulates the sarcomere lattice and stabilizes the OFF states of myosin heads

Muscle contraction is produced via the interaction of myofilaments and is regulated so that muscle performance matches demand. Myosin-binding protein C (MyBP-C) is a long and flexible protein that is tightly bound to the thick filament at its C-terminal end (MyBP-C C8C10 ), but may be loosely bound at its middle- and N-terminal end (MyBP-C C1C7 ) to myosin heads and/or the thin filament. MyBP-C is thought to control muscle contraction via the regulation of myosin motors, as mutations lead to debilitating disease. We use a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-C C1C7 domains of fast MyBP-C in permeabilized skeletal muscle. We show that cleavage leads to alterations in crossbridge kinetics and passive structural signatures of myofilaments that are indicative of a shift of myosin heads towards the ON state, highlighting the importance of MyBP-C C1C7 to myofilament force production and regulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Programmed cell death regulator BAP2 is required for IRE1-mediated unfolded protein response in Arabidopsis

Environmental and physiological situations can challenge the balance between protein synthesis and folding capacity of the endoplasmic reticulum (ER) and cause ER stress, a potentially lethal condition. The unfolded protein response (UPR) restores ER homeostasis or actuates programmed cell death (PCD) when ER stress is unresolved. The cell fate determination mechanisms of the UPR are not well understood, especially in plants. Here, we integrate genetics and ER stress profiling with natural variation and quantitative trait locus analysis of 350 natural accessions of the model species Arabidopsis thaliana . Our analyses implicate a single nucleotide polymorphism to the loss of function of the general PCD regulator BON-ASSOCIATED PROTEIN2 (BAP2) in UPR outcomes. We establish that ER stress-induced BAP2 expression is antagonistically regulated by the UPR master regulator, inositol-requiring enzyme 1 (IRE1), and that BAP2 controls adaptive UPR amplitude in ER stress and ignites pro-death mechanisms in conditions of UPR insufficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

HURP regulates Kif18A recruitment and activity to synergistically control microtubule dynamics

Abstract During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determine the binding mode of HURP to microtubules using cryo-EM. The structure helps rationalize why HURP functions as a microtubule stabilizer. Additionally, HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observe that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in microtubule length control.

Science & Technology - Other Topics↗
DOE OSTI2025

Molecular basis for human respiratory syncytial virus transcriptional regulator NS1 interactions with MED25

The Mediator complex facilitates interactions between transcription factors and RNA polymerase II, a process that is required for host gene transcription, including in response to viral infections. Among the many subunits in the Mediator complex, the MED25 subunit has been shown to be a target for viral activators during infection. Here we provide the molecular basis for the interaction between human respiratory syncytial virus (hRSV) nonstructural 1 protein (NS1) and the activator interaction domain (ACID) of MED25. The X-ray crystal structure of the complex revealed that NS1 straddles and binds two faces of MED25 ACID. This interaction is distinct from previously known viral activators. Importantly, our data support the conformational flexibility of viral transcriptional regulators. Furthermore, ChIP-seq and RNA-seq analysis identified the ATF3 transcription factor and a role for NS1/Mediator/ATF3 interaction in host gene regulation in hRSV infections. Our findings provide a molecular basis for hRSV NS1-based regulation of host gene transcription and reveal how viruses exploit the conformational heterogeneity at fuzzy transcription activator interfaces.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Gibberellins promote polar auxin transport to regulate stem cell fate decisions in cambium

Vascular cambium contains bifacial stem cells, which produce secondary xylem to one side and secondary phloem to the other. However, how these fate decisions are regulated is unknown. Here we show that the positioning of an auxin signalling maximum within the cambium determines the fate of stem cell daughters. The position is modulated by gibberellin-regulated, PIN1-dependent polar auxin transport. Gibberellin treatment broadens auxin maximum from the xylem side of the cambium towards the phloem. As a result, xylem-side stem cell daughter preferentially differentiates into xylem, while phloem-side daughter retains stem cell identity. Occasionally, this broadening leads to direct specification of both daughters as xylem, and consequently, adjacent phloem-identity cell reverts to being stem cell. Conversely, reduced gibberellin levels favour specification of phloem-side stem cell daughter as phloem. Together, our data provide a mechanism by which gibberellin regulates the ratio of xylem and phloem production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Dynamics of ER stress-induced gene regulation in plants

Endoplasmic reticulum (ER) stress is a potentially lethal condition that is induced by the abnormal accumulation of unfolded or misfolded secretory proteins in the ER. In eukaryotes, ER stress is managed by the unfolded protein response (UPR) through a tightly regulated, yet highly dynamic, reprogramming of gene transcription. Although the core principles of the UPR are similar across eukaryotes, unique features of the plant UPR reflect the adaptability of plants to their ever-changing environments and the need to balance the demands of growth and development with the response to environmental stressors. Here, the past decades have seen notable progress in understanding the mechanisms underlying ER stress sensing and signaling transduction pathways, implicating the UPR in the effects of physiological and induced ER stress on plant growth and crop yield. Facilitated by sequencing technologies and advances in genetic and genomic resources, recent efforts have driven the discovery of transcriptional regulators and elucidated the mechanisms that mediate the dynamic and precise gene regulation in response to ER stress at the systems level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Complex regulation in a Comamonas platform for diverse aromatic carbon metabolism

Critical to a sustainable energy future are microbial platforms that can process aromatic carbons from the largely untapped reservoir of lignin and plastic feedstocks. Comamonas species present promising bacterial candidates for such platforms because they can use a range of natural and xenobiotic aromatic compounds and often possess innate genetic constraints that avoid competition with sugars. However, the metabolic reactions of these species are underexplored, and the regulatory mechanisms are unknown. Here we identify multilevel regulation in the conversion of lignin-related natural aromatic compounds, 4-hydroxybenzoate and vanillate, and the plastics-related xenobiotic aromatic compound, terephthalate, in Comamonas testosteroni KF-1. Transcription-level regulation controls initial catabolism and cleavage, but metabolite-level thermodynamic regulation governs fluxes in central carbon metabolism. Quantitative 13 C mapping of tricarboxylic acid cycle and cataplerotic reactions elucidates key carbon routing not evident from enzyme abundance changes. This scheme of transcriptional activation coupled with metabolic fine-tuning challenges outcome predictions during metabolic manipulations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2022

Deciphering the transcriptional regulation of the catabolism of lignin-derived aromatics in Rhodococcus opacus PD630

Abstract Rhodococcus opacus PD630 has considerable potential as a platform for valorizing lignin due to its innate “biological funneling” pathways. However, the transcriptional regulation of the aromatic catabolic pathways and the mechanisms controlling aromatic catabolic operons in response to different aromatic mixtures are still underexplored. Here, we identified and studied the transcription factors for aromatic degradation using GFP-based sensors and comprehensive deletion analyses. Our results demonstrate that the funneling pathways for phenol, guaiacol, 4-hydroxybenzoate, and vanillate are controlled by transcriptional activators. The two different branches of the β-ketoadipate pathway, however, are controlled by transcriptional repressors. Additionally, promoter activity assays revealed that the substrate hierarchy in R. opacus may be ascribed to the transcriptional cross-regulation of the individual aromatic funneling pathways. These results provide clues to clarify the molecule-level mechanisms underlying the complex regulation of aromatic catabolism, which facilitates the development of R. opacus as a promising chassis for valorizing lignin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

The fluctuation-dissipation theorem for stochastic kinetics—Implications on genetic regulations

The Fluctuation-Dissipation theorem (FDT) connects the “memory” in the fluctuation in equilibrium to the response of a system after a perturbation, which has been a fundamental ground in many branches of physics. When viewing a cell as a stochastic biochemical system, the cell's response under a perturbation is related to its intrinsic steady-state correlation functions via the FDT, a theorem we derived and present in this work. FDT allows us to use the noise to derive dynamic response and infer dynamic properties in the system. We tested FDT's validity with gene regulation models and found that it is limited to the linear response. For an indirect regulation pathway where unknown components may exist, FDT still works within the linear response region. Thus, FDT may be used for systems with partial knowledge, and it is potentially possible to identify the existence of unobserved components. With FDT, the dynamic response can be composed of steady-state measurements without the complete detailed knowledge for the regulation or kinetics. The response function derived can give important insights into the dynamics and time scales of the system.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2025

Charge regulation effects on colloidal mixture nanoparticles

Changes in pH within a system containing dissociable sites affect the protonation and deprotonation of these groups, thereby influencing their physical properties. In response, the system modifies their surface charge, affecting electrostatic interactions, aggregation, stability, and structural behavior. Although the pH can be tuned in experiments, it is difficult to model this phenomenon using simulations or theoretical approaches. Here, we perform hybrid Monte Carlo-molecular dynamics simulations to model charge regulation effects in an equimolar colloidal charged system. We compare charge regulation effects with those of a system in which the charges of colloidal nanoparticles are not dissociable. The comparison between the two cases modifies the phase diagram, and it changes the volume fraction where a percolation network of nanoparticles is found. Charge regulation is found to destroy network formation, as the charge in the nanoparticles is modified because of the cooperativity dependency of the degree of charge dissociation sites among the nanoparticles favoring cluster formation. Furthermore, our work suggests that the ionic and/or electronic conductivity in functionalized nanoparticles can be modified by changing pH values. It also guides the experimental design of oppositely charged nanoparticles as inks for 3D printing processes.

Classical statistical mechanics↗
DOE OSTI2015

Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa

Significance Pseudomonas aeruginosa , an opportunistic pathogen that can cause fatal chronic infections, relies on the intracellular second-messenger c-di-GMP to form robust multicellular biofilms during host tissue colonization. c-di-GMP is sensed directly by the transcription regulator FleQ, which inversely regulates flagellar motility and exopolysaccharide secretion to secure a planktonic to sessile life-form transition. FleQ belongs to the diverse family of AAA+ ATPase enhancer-binding proteins, but how its noncanonical function on transcriptional regulation is controlled by c-di-GMP remains enigmatic. Here, we report structural and functional data that identify an unusual mode of c-di-GMP recognition accompanied by a major quaternary structure reorganization. Our analyses offer a consensus to previous studies and unique insights into the mechanism of action of FleQ and FleQ-like proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Evidence for a cysteine-mediated mechanism of excitation energy regulation in a photosynthetic antenna complex

Significance All photosynthetic organisms face the challenge of absorbing solar energy and regulating its flow through their light-harvesting antennas across widely varying photic conditions. For anoxygenic phototrophs, this process is complicated by the need to downregulate photosynthetic output when oxygen is encountered. The Fenna–Matthews–Olson protein from green sulfur bacteria is able to quench excitations in aerobic conditions effectively despite its apparent lack of photoprotective accessory molecules, indicating a previously unidentified type of energy transfer regulation. In this study, we provide evidence for a novel energy-quenching mechanism involving cysteine–bacteriochlorophyll photochemistry. This interaction should be able to be programed into other natural or bio-inspired antennas, opening new possibilities for regulating these systems in response to excess light.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa

Significance Wood is a widely used renewable feedstock for industrial production and energy generation. The secondary cell wall (SCW) is the major component of wood. Two key transcription factor families, Vascular-Related NAC-Domain (VND) and Secondary Wall-Associated NAC Domain (SND), are master gene regulators for SCW biosynthesis. However, plants exhibit stunted growth or abnormal SCW development under excess VND or SND gene expression. In this study, we show that two splice variants, PtrVND6-C1 IR and PtrSND1-A2 IR , each from VND and SND families, act as negative regulators. We propose that PtrVND6-C1 IR and PtrSND1-A2 IR function together for reciprocal cross-regulation of VND and SND families to maintain homeostasis for xylem differentiation and plant development.

Lin, Ying-Chung Jimmy↗
DOE OSTI2018

Multiresponsive polymeric microstructures with encoded predetermined and self-regulated deformability

Dynamic functions of biological organisms often rely on arrays of actively deformable microstructures undergoing a nearly unlimited repertoire of predetermined and self-regulated reconfigurations and motions, most of which are difficult or not yet possible to achieve in synthetic systems. Here, we introduce stimuli-responsive microstructures based on liquid-crystalline elastomers (LCEs) that display a broad range of hierarchical, even mechanically unfavored deformation behaviors. By polymerizing molded prepolymer in patterned magnetic fields, we encode any desired uniform mesogen orientation into the resulting LCE microstructures, which is then read out upon heating above the nematic–isotropic transition temperature (T N–I ) as a specific prescribed deformation, such as twisting, in- and out-of-plane tilting, stretching, or contraction. By further introducing light-responsive moieties, we demonstrate unique multifunctionality of the LCEs capable of three actuation modes: self-regulated bending toward the light source at T < T N–I , magnetic-field–encoded predetermined deformation at T > T N–I , and direction-dependent self-regulated motion toward the light at T > T N–I . We develop approaches to create patterned arrays of microstructures with encoded multiple area-specific deformation modes and show their functions in responsive release of cargo, image concealment, and light-controlled reflectivity. We foresee that this platform can be widely applied in switchable adhesion, information encryption, autonomous antennae, energy harvesting, soft robotics, and smart buildings.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

GUN1 interacts with MORF2 to regulate plastid RNA editing during retrograde signaling

During development or under stress, chloroplasts generate signals that regulate the expression of a large number of nuclear genes, a process called retrograde signaling. GENOMES UNCOUPLED 1 (GUN1) is an important regulator of this pathway. In this study, we have discovered an unexpected role for GUN1 in plastid RNA editing, as gun1 mutations affect RNA-editing efficiency at multiple sites in plastids during retrograde signaling. GUN1 plays a direct role in RNA editing by physically interacting with MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2). MORF2 overexpression causes widespread RNA-editing changes and a strong genomes uncoupled ( gun ) molecular phenotype similar to gun1 . MORF2 further interacts with RNA-editing site-specificity factors: ORGANELLE TRANSCRIPT PROCESSING 81 (OTP81), ORGANELLE TRANSCRIPT PROCESSING 84 (OTP84), and YELLOW SEEDLINGS 1 (YS1). We further show that otp81 , otp84 , and ys1 single mutants each exhibit a very weak gun phenotype, but combining the three mutations enhances the phenotype. Our study uncovers a role for GUN1 in the regulation of RNA-editing efficiency in damaged chloroplasts and suggests that MORF2 is involved in retrograde signaling.

59 BASIC BIOLOGICAL SCIENCES↗