Biology subjects

Williamson, J. R.

Publications and source records attributed to Williamson, J. R..

12 recordsLinked to original sources

bioRxiv2026

ESCAPE: assigning site-specific activity to covalent ligands in cells by prime editing

Chemical proteomics has identified covalent ligands targeting cysteine residues across many hundreds of human proteins. The functional effects of these liganding events, however, remain challenging to assign at scale. Here we describe ESCAPE (Endogenous Site-specific Competition Assays using Prime Editors), a platform for the site-resolved functional analysis of covalent ligands in cells. In this method, cysteine-to-serine substitutions are generated by prime editing to abrogate covalent ligand-protein interactions, and the impact of these edits on ligand-induced cellular phenotypes is quantified through allele frequency-based resistance scores. Applied to ligandable cysteines mapped by activity-based protein profiling in 50+ proteins, ESCAPE identified multiple covalent ligand-protein interactions that impair cancer cell growth, including azetidine butynamides that target a non-orthosteric cysteine in the RNA helicase DDX49 to disrupt 18S rRNA processing, 40S ribosome assembly, and protein synthesis. ESCAPE thus provides a scalable framework for the functional characterization of covalent ligands targeting structurally and mechanistically diverse proteins.

biochemistry↗

bioRxiv2024

Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. B. subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after E. coli and T. thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

microbiology↗

bioRxiv2024

Domain consolidation in Bacterial 50S assembly revealed by Anti-Sense Oligonucleotide Probing

Investigating the intricate and rapid folding kinetics of large RNA-protein complexes (RNPs), like the bacterial ribosome, remains a formidable challenge in structural biology. Previous genetic approaches to probe assembly have focused on modulating the expression of either r-proteins or assembly factors. Here, anti-sense oligonucleotides (ASOs) were used to disrupt native RNA/RNA and RNA/protein interactions, in order to generate novel folding intermediates. In an in vitro co-transcriptional assembly assay, 8 assembly inhibitor ASOs were identified. Using cryo-electron microscopy, 38 new intermediate structures were determined resulting from the specific inhibitions. In particular a novel intermediate class provided compelling evidence of independent rRNA domain folding before proper interdomain docking. Three PNAs targeting domain-I of 23S- rRNA further subdivided the previously identified assembly core into smaller blocks representing the earliest steps in assembly. The resulting assembly graph reveals template-directed RNA foldon docking and domain consolidation, which provides a hierarchical view of the RNP assembly process.

biochemistry↗

bioRxiv2024

Assembly of the Bacterial Ribosome with Circularly Permuted rRNA

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

biophysics↗

bioRxiv2023

Proteome partitioning constraints on long-term laboratory evolution

Adaptive laboratory evolution experiments provide a controlled context in which the dynamics of selection and adaptation can be followed in real-time at the single-nucleotide level1. And yet this precision introduces hundreds of degrees-of-freedom as genetic changes accrue in parallel lineages over generations2. On short timescales, physiological constraints have been leveraged to provide a coarse-grained view of bacterial gene expression characterized by a small set of phenomenological parameters3-5. Here, we ask whether this same framework, operating at a level between genotype and fitness, informs physiological changes that occur on evolutionary timescales. Using Lenskis Ara-1 lineage adapted to growth in glucose minimal medium6, we find that the proteome is substantially remodeled over 40 000 generations. We apply our existing quantitative proteomics analysis to partition hundreds of expressed proteins into six sectors with shared metabolic function and physiological response4. To accommodate the increased growth rates in the evolved strains, expression of metabolic enzymes undergoes sector-specific adaptation to enable increased fluxes. We find that catabolic proteins adapt by increasing the total enzyme abundance, whereas anabolic and glycolytic proteins exhibit decreased free-enzyme pools. We propose that flux-dependent regulation7 and substrate saturation8 can account for the sector-specific remodeling.

microbiology↗

bioRxiv2023

Early HIV-1 Gag Assembly on Lipid Membrane with vRNA

Mass photometry (MP) was used to investigate the assembly of myristoylated full-length HIV-1 Gag (myr-Gag) and vRNA 5 UTR fragment in a supported lipid bilayer (SLB) model system. The MP trajectories demonstrated that Gag trimerization on the membrane is a key step of early Gag assembly in the presence of vRNA. Growth of myr-Gag oligomers requires vRNA, occuring by addition of 1 or 2 monomers at a time from solution. These data support a model where formation of the Gag hexamers characteristic of the immature capsid lattice occurs by a gradual edge expansion, following a trimeric nucleation event. These dynamic single molecule data involving protein, RNA, and lipid components together, provide novel and fundamental insights into the initiation of virus capsid assembly.

biophysics↗

bioRxiv2022

Unsupervised Voxel-based Segmentation reveals a Landscape of Bacterial Ribosome Large Subunit Early Assembly

Ribosome biogenesis is a complex but efficient process in rapidly growing bacteria, where assemble a functional 70S ribosome takes ~ 2 min, involving participation of 3 rRNAs, 50 r-proteins and dozens of assembly factors. In vitro reconstitution using different subsets of large subunit (50S, LSU) proteins with rRNAs, pioneered by Nierhaus lab, resulted in the Nierhaus assembly map, embodying the cooperativity and dependency for binding of LSU r-proteins to 23S rRNA. Critically absent from the Nierhaus map is the underlying folding of the rRNA that creates the binding sites for the r-proteins. In addition, the relationship of the observed cooperativity in vitro to the co-transcriptional assembly in cells remains to be determined. Pre-50S intermediates accumulate at low temperature in {Delta}deaD, a DEAD-box helicase implicated in 50S assembly. We solved 21 pre-50S density maps from intermediate-containing fractions using cryo-EM. In the newly solved maps, we discovered the earliest intermediate ever reported, consisting of domain I at the 5-end of 23S rRNA. To probe the mechanism behind the maps during assembly, we developed a novel density map segmentation and dependency analysis method. Ten cooperative assembly blocks were identified from segmentation of the maps, and these were organized into a block dependency map. This is the first time the dependencies on folding of rRNA helices and ribosomal protein binding could be integrated into a complete assembly map. In addition, we showed how the exit tunnel is folded on the solvent side, serving as a scaffold for 50S maturation. Using this new segmentation analysis method, we revisited previously reported bL17-depletion and {Delta}srmB datasets. Most remarkably, the other two datasets are also consistent with the block dependency, implying a unified early assembly pathway and flexible maturation landscape in early 50S biogenesis.

biochemistry↗

bioRxiv2022

Near-Physiological in vitro Assembly of 50S Ribosomes Involves Parallel Pathways

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as an ideal model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the whole assembly process were resolved using cryo-EM single particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported up to now, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following a defined set of dependencies, revealing the parallel assembly pathways at both early and late assembly stages of the 50S subunit.

biochemistry↗

bioRxiv2022

A Metabolic Sum Rule Dictates Bacterial Response to Short-Chain Fatty Acid Stress

Short-chain fatty acids (SCFAs) such as acetate accumulate in fermentative environments, inhibiting many types of bacteria. While it is known that cells accumulate SCFAs to high concentrations internally, the cause of SCFA toxicity is not understood. By forcing Escherichia coli cells to accumulate a variety of "useless metabolites", we establish via extensive omic analysis a metabolic sum rule, by which the accumulation of exogenous metabolites such as acetate forces the depletion of endogenous metabolites. The latter leads to bottlenecks in biosynthesis, manifested as a simple linear relation between useless metabolite accumulation and growth reduction. Guided by quantitative models, we show that acetate-stressed cells optimize growth by partially acidifying their own cytoplasm, which reduces acetate accumulation, restoring the endogenous metabolites as allowed by the sum rule.

microbiology↗

bioRxiv2022

Pre-B cell receptor acts as a selectivity switch for Galectin-1 at the pre-B cell surface

Galectins are glycan binding proteins translating the sugar-encoded information of cellular glycoconjugates into many physiological activities including immunity, cell migration, and signaling. During early B lymphocytes (BL) development at the pre-B cell stage, BL express the pre-B cell receptor (pre-BCR) and are supported by mesenchymal stromal cells secreting Galectin-1 (Gal-1). Gal-1 interacts with glycosylated receptors from stromal and pre-B cell surfaces but also with the pre-BCR through a direct carbohydrate-independent contact. How this interaction might interplay with the glycan-decoding function of Gal-1 is unknown. Here, we investigated Gal-1 binding to cell surface ligands using NMR spectroscopy on native membranes. We showed that pre-BCR regulates Gal-1 binding to specifically target 2,3-sialylated receptors on pre-B cells. Upon pre-BCR interaction, dynamic changes resulted in additional contacts with 2,3-sialylated glycans converting Gal-1 from an exo- to an endo-type lectin. Remarkably, this selectivity switch is able to promote pre-B cell survival. Altogether, we shed light on a new mechanism allowing fine-tuning of Galectin specificity at the cell surfaces.

biochemistry↗

bioRxiv2021

Quantitative Mining of Compositional Heterogeneity in Cryo-EM Datasets of Ribosome Assembly Intermediates

Macromolecular complexes are dynamic entities whose function is often intertwined with their many structural configurations. Single particle cryo-electron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct populations into different groups through computational classification. However, current workflows are limited, and there is a dearth of tools for surveying the heterogeneity landscape, quantitatively analyzing heterogeneous particle populations after classification, deciding how many unique classes are represented by the data, and accurately cross-comparing reconstructions. Here, we develop a workflow that contains discovery and analysis modules to quantitatively mine cryo-EM data for a set of structures with maximal diversity. This workflow was applied to a dataset of E. coli 50S ribosome assembly intermediates, which is characterized by significant structural heterogeneity. We identified new branch points in the assembly process and characterized the interactions of an assembly factor with immature intermediates. While the tools described here were developed for ribosome assembly, they should be broadly applicable to the analysis of other heterogeneous cryo-EM datasets.

biophysics↗

bioRxiv2018

Translation Attenuation by Minocycline Increases Lifespan and Improves Proteostasis in Old Post-Stress-Responsive Organisms

Aging impairs the activation of Stress Signaling Pathways (SSPs), preventing the induction of longevity mechanisms late in life. Here we show that the antibiotic minocycline increases lifespan and reduces protein aggregation even in old, SSP-deficient C. elegans by targeting cytoplasmic ribosomes, preferentially attenuating translation of highly translated mRNAs. In contrast to most other longevity paradigms, minocycline inhibits rather than activates all major SSPs and extends lifespan in mutants deficient in the activation of SSPs, lysosomal or autophagic pathways. We propose that minocycline lowers the concentration of newly synthesized aggregation-prone proteins, resulting in a relative increase in protein-folding capacity without the necessity to induce protein-folding pathways. Our study suggests that in old individuals with incapacitated SSPs or autophagic pathways, pharmacological attenuation of cytoplasmic translation is a promising strategy to reduce protein aggregation. Altogether, it provides a geroprotecive mechanism for the many beneficial effects of tetracyclines in models of neurodegenerative disease.

physiology↗