Biology subjects

Cate, J. H. D.

Publications and source records attributed to Cate, J. H. D..

29 records · Page 2Linked to original sources

bioRxiv2022

Candidatus Nealsonbacteria (OD1) in a methanogenic benzene-degrading enrichment culture is likely an ectosymbiotic biomass recycler

The Candidate Phyla Radiation (CPR) is a very large group of bacteria with no pure culture representatives, first discovered by metagenomic analyses. Within the CPR, candidate phylum Parcubacteria (previously referred to as OD1) within the candidate superphylum Patescibacteria is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the Candidate clade Nealsonbacteria. Because of its persistence over many years, we hypothesized that Ca. Nealsonbacteria DGGOD1a must serve an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10 fold) increase in the absolute abundance of Ca. Nealsonbacteria DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate Ca. Nealsonbacteria in biomass recycling. Fluorescent in situ hybridization and cryogenic transmission electron microscope images revealed that Ca. Nealsonbacteria DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other Ca. Nealsonbacteria found in anoxic environments.

microbiology↗

bioRxiv2020

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Here we report a protocol for isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and prior to transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

plant biology↗

bioRxiv2020

Structure of the Bacterial Ribosome at 2 A Resolution

Continuing advances in cryo-electron microscopy (cryo-EM) demonstrate the promise it holds for revealing biological structures at chemical resolution, in which noncovalent interactions, RNA and protein modifications, and solvation can be modeled accurately. At present, the best cryo-EM-derived models of the bacterial ribosome are of the large (50S) ribosomal subunit with effective global resolutions of 2.4-2.5 [A], based on map-to-model Fourier shell correlation (FSC). Here we present a model of the E. coli 70S ribosome with an effective global resolution of 2.0 [A], based on maps showcasing unambiguous positioning of residues, their detailed chemical interactions, and chemical modifications. These modifications include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former of which is likely conserved in all domains of life. The model also defines extensive solvation of the small (30S) ribosomal subunit for the first time, as well as interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The high quality of the maps now allows a deeper phylogenetic analysis of ribosomal components, and identification of structural conservation to the level of solvation. The maps and models of the bacterial ribosome presented here should enable future structural analysis of the chemical basis for translation, and the development of robust tools for cryo-EM structure modeling and refinement.

biophysics↗

bioRxiv2020

Selective inhibition of human translation by a drug-like compound that traps terminated protein nascent chains on the ribosome

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation 1-4. These molecules also inhibit translation termination 4 by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 arrests translation at the stop codon by slowing hydrolysis of the protein nascent chain (NC) from peptidyl-site (P-site) tRNA by eukaryotic release factor 1 (eRF1). After NC hydrolysis from the P-site tRNA, PF846 traps the NC in the ribosome exit tunnel in a compact -helical conformation that induces 28S rRNA nucleotide rearrangements propagating back to the ribosome peptidyl transferase center (PTC). Mutational analyses and human cell-based experiments elucidate the pivotal amino acids of the NC required for PF846-dependent termination arrest, all of which face the PF846 side of the ribosome exit tunnel. The structural and functional data support a model in which PF846 inhibits translation termination by inducing allosteric conformational rearrangements in the NC and PTC that suppress peptidyl-tRNA hydrolysis promoted by eRF1, and trap the NC in the ribosome exit tunnel. This unprecedented mechanism of action reveals new principles of translation termination and lays the foundation for new therapeutic strategies.

biophysics↗

bioRxiv2019

Dynamics of T cell activation mediated by eIF3 interactions with T cell receptor mRNAs

Activation of T cells requires a global surge in cellular protein synthesis, accompanied by a large increase in translation initiation1-4. A central component of the translation initiation machinery-the multi-subunit eukaryotic initiation factor 3 (eIF3)-is rapidly turned on when quiescent T cells are stimulated3. However, the precise role eIF3 plays in activated T cells is not known. Using a global transcriptome crosslinking approach, we show human eIF3 interacts with a distinct set of mRNAs in activated Jurkat cells. A subset of these mRNAs, including those encoding the T cell receptor (TCR) subunits TCRA and TCRB, crosslink to eIF3 across the entire length of the mRNA. The TCRA and TCRB mRNAs do not co-localize with translationally repressed environments of P-bodies or stress granules but form distinct granules, potentially acting as translation "hot-spots." T cell activation through CD28 causes a burst of TCR translation controlled by elements in the 3-untranslated regions (3-UTRs) of the TCRA and TCRB mRNAs that directly contact eIF3 and that are required for T cell activity. These results highlight a new role for eIF3 in regulating the translation dynamics of the TCR and provide insights that can guide the engineering of T cells used in cell immunotherapy applications.

molecular biology↗

bioRxiv2019

Defects in the assembly of ribosomes selected for β-amino acid incorporation

Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several non-standard monomers including D-amino acids, dipeptides, and {beta}-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo {beta}-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering towards new catalytic abilities.

biochemistry↗

bioRxiv2019

Phazolicin - a Novel Thiazole/Oxazole-Modified Peptide Inhibiting the Bacterial Ribosome in a Species-Specific Way.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding and largely untapped class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display an outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium sp. Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants belonging to the Rhizobiales. PHZ inhibits prokaryotic translation through the obstruction of the passage of the nascent peptide through the ribosome exit channel. The cryo-EM structure of the Escherichia coli ribosome with bound PHZ revealed that the drug interacts with the 23S rRNA and ribosomal proteins uL4 and uL22 and obstructs the exit tunnel in a way that is distinct from other compounds blocking the exit channel. We show that the sequence of uL4 ribosomal protein loop involved in PHZ binding determines the species-specificity of antibiotic interaction with its target. PHZ and its predicted homologs from other bacterial species expand the known diversity of LAPs and may be used in the future as biocontrol agents for the needs of agriculture.

molecular biology↗

bioRxiv2018

Differences in the path to exit the ribosome across the three domains of life

Recent advances in biological imaging have led to a surge of fine-resolution structures of the ribosome from diverse organisms. Comparing these structures, especially the exit tunnel, to characterize the key similarities and differences across species is essential for various important applications, such as designing antibiotic drugs and understanding the intricate details of translation dynamics. Here, we compile and compare 20 fine-resolution cryo-EM and X-ray crystallography structures of the ribosome recently obtained from all three domains of life (bacteria, archaea and eukarya). We first show that a hierarchical clustering of tunnel shapes closely reflects the species phylogeny. Then, by analyzing the ribosomal RNAs and proteins localized near the tunnel, we explain the observed geometric variations and show direct association between the conservations of the geometry, structure, and sequence. We find that the tunnel is more conserved in its upper part, from the polypeptide transferase center to the constriction site. In the lower part, tunnels are significantly narrower in eukaryotes than in bacteria, and we provide evidence for the existence of a second constriction site in eukaryotic tunnels. We also show that ribosomal RNA and protein sequences are more likely to be conserved closer to the tunnel, as is the presence of positively charged amino acids. Overall, our comparative analysis shows how the geometric and biophysical properties of the exit tunnel play an important role in ensuring proper transit of the nascent polypeptide chain, and may explain the differences observed in several co-translational processes across species.

biophysics↗

bioRxiv2018

Modulating long-range energetics via helix stabilization: a case study using T4 lysozyme

Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the C-terminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization.

biophysics↗

bioRxiv2017

Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

synthetic biology↗

bioRxiv2016

Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5 [A] or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5 [A], while the resolution of the map reached 4.0 [A] for the same number of particles, when the estimated resolution of streptavidin crystal was 4 [A] or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6 [A], a marked improvement over the value of 3.9 [A] obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.

biophysics↗