Biology subjects

Shulgina, Y.

Publications and source records attributed to Shulgina, Y..

10 recordsLinked to original sources

bioRxiv2026

tRNA-derived second messengers confer bacterial immunity

Bacterial small-molecule messengers that function in diverse immune signaling pathways have been thought to derive strictly from biosynthesis using nucleotide and cofactor substrates. Here we report a nuclease-driven immune signaling pathway in which tRNA cleavage generates a specific small molecule that triggers cell death in response to RNA-guided transcript recognition in prokaryotes. We identify a class of type VI-B CRISPR systems in which the RNA-guided nuclease Cas13 and its accessory transmembrane protein, 2TM{beta}, are co-encoded in an operon and are jointly required for bacteriophage defense. Upon target RNA recognition, Cas13 activates 2TM{beta} by producing mono- and dinucleotide messengers bearing 2',3'-cyclic uridine phosphate ends by tRNA cleavage. Activated 2TM{beta} destroys infected cells by forming a multimeric complex that disrupts cell membranes to mediate cell death. These findings demonstrate RNA-programmed tRNA cleavage for second-messenger biogenesis, revealing a new logic for prokaryotic immune signaling.

microbiology↗

bioRxiv2026

Enzymatic formation of a conserved isoaspartate in ribosomal protein uS11

Isoaspartate (isoAsp) formation is typically viewed as a "molecular clock" through nonenzymatic degradation of aspartate or asparagine during protein aging. Here we report a nearly universal enzymatic pathway for the formation of a conserved isoAsp in the bacterial ribosomal protein uS11. Proteome-wide protein-protein interaction scans using AlphaFold3 identified YbeY as a candidate enzyme from Escherichia coli. NMR spectroscopy supported a stable YbeY-uS11 complex from Thermotoga maritima. Biochemical assays indicated that EcYbeY catalysis is zinc-dependent and prefers the conserved Asn-Gly motif for isoAsp formation. A high-resolution cryo-electron microscopy structure of the 70S ribosome from E. coli {Delta}ybeY revealed that loss of isoAsp alters contacts with the 16S rRNA groove and bS21. Phylogenetic analysis indicated that YbeY is present in almost all bacteria, and its absence is correlated to changes in the Asn-Gly motif of uS11. Additionally, our structural analyses implicate Fap7 as the functional counterpart in archaea and eukaryotes.

biochemistry↗

bioRxiv2026

A threonine-to-aspartate ACA codon reassignment in uncultivated Acidimicrobiales bacteria

The list of alternative genetic codes continues to grow, yet sense-to-sense codon reassignments remain very rare. Here we analyze a new sense-to-sense codon reassignment in bacteria, where the ACA codon, which canonically specifies threonine, has been predicted to encode aspartate. This so far unprecedented genetic code alteration is confined to a clade within the RAAP-2 family of the order Acidimicrobiales (phylum Actinomycetota). Validated by multiple alignments of highly conserved proteins, the reassignment is further supported by the presence of a tRNAUGU that lacks the G1:C72 threonylation identity element, suggesting that this tRNA is charged with aspartate instead of threonine. Our findings not only expand the set of known natural departures from the canonical genetic code by a novel type of sense-to-sense reassignment but also implicate the ACA codon that was so far considered invariably conserved in its meaning.

genomics↗

bioRxiv2026

A family of archaeal hibernation factors that bind in tandem and protect ribosomes in dormant cells

Under nutrient limitation or stress, ribosome hibernation factors inactivate and protect ribosomes. Although ribosome hibernation plays an important role in microbes, we lack a complete understanding of this process in archaea. Here, we identify a family of hibernation factors, which we designate as single ribosomal subunit inhibitors (SriA-SriD), from the methanogenic archaeon Methanosarcina acetivorans. All four sri genes are encoded in an operon and each Sri protein inhibits protein synthesis in vitro. Deletion of sri genes in M. acetivorans impaired growth recovery after prolonged stationary phase and also led to depletion of the small ribosomal subunit. Cryo-EM structures show that Sri proteins bind to the ribosome in tandem and form conserved protein-protein interfaces. Sri is broadly distributed across archaeal phyla and sri genes frequently co-occur. Together, these findings establish Sri proteins as a distinct group of hibernation factors that protect ribosomes during dormancy and expand our understanding of ribosome hibernation in archaea.

microbiology↗

bioRxiv2024

Structure of an Archaeal Ribosome with a Divergent Active Site

The ribosome is the universal translator of the genetic code and is shared across all life. Despite divergence in ribosome structure over the course of evolution, the peptidyl transferase center (PTC), the catalytic site of the ribosome, has been thought to be nearly universally conserved. Here, we identify clades of archaea that have highly divergent ribosomal RNA sequences in the PTC. To understand how these PTC sequences fold, we determined cryo-EM structures of the Pyrobaculum calidifontis ribosome. We find that sequence variation leads to the rearrangement of key PTC base triples and differences between archaeal and bacterial ribosomal proteins also enable sequence variation in archaeal PTCs. Finally, we identify a novel archaeal ribosome hibernation factor that differs from known bacterial and eukaryotic hibernation factors and is found in multiple archaeal phyla. Overall, this work identifies factors that regulate ribosome function in archaea and reveals a larger diversity of the most ancient sequences in the ribosome.

evolutionary biology↗

bioRxiv2024

RNA language models predict mutations that improve RNA function

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data1. Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function2-4. We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB)5. GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which overlapping triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB- derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

synthetic biology↗

bioRxiv2023

How did UGA codon translation as tryptophan evolve in certain ciliates? A critique of Kachale et al. 2023 Nature

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition1. All non-standard ciliate genetic codes involve stop codon reassignments1,2,3. Two of these codes are ambiguous1-3, with "stop" codons either translated or terminating translation depending on their context2,3. Ambiguous genetic codes have arisen not only in ciliates, but also independently in trypanosomatids from the genus Blastocrithidia4 and an alveolate species from the genus Amoebophrya5. Two ambiguous genetic codes in ciliates share translation of UGA "stop" codons as tryptophan with Blastocrithidia and the Amoebophrya species. tRNA genes with complementary anticodons to reassigned UAA and UAG stop codons have invariably been found in ciliate species that translate these codons1,2. Furthermore, though a UGA-cognate tRNACysUCA was reported in Euplotes6, a ciliate genus that translates UGA as cysteine, vexingly, no nuclear genome-encoded tRNATrpUCA has been found in ciliate species with UGA tryptophan codons. Recently, Kachale et al. provided evidence for UGA translation as tryptophan in Blastocrithidia nonstop and the ciliate Condylostoma magnum using 4 base pair anticodon stem (AS) near-cognate tryptophan tRNATrpCCAs, rather than the typical 5 base pair stem tRNAs7. New tRNA data we report from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised that a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), favours translation of UGA as tryptophan instead of termination7. Contrary to Kachale et al, we propose such substitutions favouring reduced eRF1 competition enhancing "stop" codon translation do not need to occur concomitantly with tRNA alterations or acquisitions to evolve new genetic codes via stop codon reassignment. We report multiple instances of the substitution investigated in Kachale et al. 2023 that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediate hypothesis for genetic code evolution, experimental evidence and our observations suggest continued potential ciliate eRF1-tRNA competition.

evolutionary biology↗

bioRxiv2021

Idiosyncratic epistasis leads to global fitness-correlated trends

Epistasis can dramatically affect evolutionary trajectories. In recent decades, protein-level fitness landscapes have revealed extensive idiosyncratic epistasis among specific mutations. In contrast, other work has found ubiquitous and apparently non-specific patterns of global diminishing-returns and increasing-costs epistasis among mutations across the genome. Here, we use a hierarchical CRISPR gene drive system to construct all combinations of 10 missense mutations from across the genome in budding yeast, and measure their fitness in six environments. We show that the resulting fitness landscapes exhibit global fitness-correlated trends, but that these trends emerge from specific idiosyncratic interactions. This provides the first experimental validation of recent theoretical work that has argued that fitness-correlated trends can emerge as the generic consequence of idiosyncratic epistasis. One-Sentence SummaryA genome-spanning fitness landscape reveals how idiosyncratic genetic interactions lead to global epistatic patterns.

evolutionary biology↗

bioRxiv2021

A computational screen for alternative genetic codes in over 250,000 genomes

The genetic code has been proposed to be a "frozen accident", but the discovery of alternative genetic codes over the past four decades has shown that it can evolve to some degree. Since most examples were found anecdotally, it is difficult to draw general conclusions about the evolutionary trajectories of codon reassignment and why some codons are affected more frequently. To fill in the diversity of genetic codes, we developed Codetta, a computational method to predict the amino acid decoding of each codon from nucleotide sequence data. We surveyed the genetic code usage of over 250,000 bacterial and archaeal genome sequences in GenBank and discovered five new reassignments of arginine codons (AGG, CGA, and CGG), representing the first sense codon changes in bacteria. In a clade of uncultivated Bacilli, the reassignment of AGG to become the dominant methionine codon likely evolved by a change in the amino acid charging of an arginine tRNA. The reassignments of CGA and/or CGG were found in genomes with low GC content, an evolutionary force which likely helped drive these codons to low frequency and enable their reassignment.

genomics↗

bioRxiv2018

Dissecting the sharp response of a canonical developmental enhancer reveals multiple sources of cooperativity

Developmental enhancers integrate graded concentrations of input transcription factors (TFs) to create sharp gene expression boundaries. Here we examine the hunchback P2 (HbP2) enhancer which drives a sharp expression pattern in the Drosophila blastoderm embryo in response to the transcriptional activator Bicoid (Bcd). We systematically interrogate cis and transfactors that influence the shape and position of expression driven by HbP2, and find that the prevailing model, based on cooperative binding of Bcd to HbP2 is not adequate. We demonstrate that other proteins, such as pioneer factors, mediator and histone modifiers influence the shape and position of the HbP2 expression pattern. By comparing our results to theory, we assess how higher-order cooperativity and energy expenditure impact boundary location and sharpness. Our results emphasize that the bacterial view of transcription regulation, where pairwise interactions between regulatory proteins dominate, must be re-examined in animals, where multiple molecular mechanisms collaborate to shape the gene regulatory function.

genetics↗