Biology subjects

Wilkinson, M. E.

Publications and source records attributed to Wilkinson, M. E..

9 recordsLinked to original sources

bioRxiv2026

Phage terminase recognition by the bacterial immune sensors Avs2 and Upx

Prokaryotes employ diverse defense strategies to detect and halt the progression of phage infection. Multiple defense systems sense phage proteins through direct binding, including antiviral STAND NTPases (Avs), which oligomerize upon target recognition to induce programmed cell death. The widespread Avs2 family was previously shown to detect the large terminase subunit of tailed phages, but the mechanism of terminase sensing was unknown. Here, we determine the structural basis of terminase recognition by Avs2 from Escherichia coli (EcAvs2). A cryo-EM structure at 2.3 [A] resolution reveals that EcAvs2 forms a flat, C4-symmetric tetramer in which each protomer is bound to a single terminase monomer. Terminase recognition is mediated by a large, shape complementary binding pocket in the EcAvs2 sensor domain, including specific contacts with an unexpected ATP molecule at the interface of EcAvs2 and terminase. Furthermore, we demonstrate that the defense protein Upx also recognizes diverse phage terminases, despite lacking sequence and structural homology to Avs. AlphaFold 3 models indicate that Upx binds an unfolded state of the core terminase ATPase domain, mediated by {beta}-augmentation. These findings highlight the distinct modes of terminase recognition across structurally diverse defense proteins.

molecular biology↗

bioRxiv2026

Host exonuclease SbcB and a phage-encoded SSB-like protein control activation of the DRT10 reverse transcriptase defense system

Defense-associated reverse transcriptases (DRTs) employ diverse mechanisms of cDNA synthesis to protect bacteria against phage infection, yet their full diversity and regulatory logic remain poorly understood. Here we provide a mechanistic characterization of UG17 (DRT10), a class-2 system within the UG/Abi reverse transcriptase lineage, classifying it into three phylogenetically and architecturally distinct subtypes with subtype-specific ncRNAs and experimentally validating its role in phage defense. DRT10 operates as a tripartite module, comprising a structured ncRNA, a SLATT effector, and an RT that catalyzes processive synthesis of DNA containing 7 nt tandem repeats. The tandem-repeat cDNA intermediate accumulates as both first- and second-strand species. SbcB suppresses DRT10-mediated toxicity and appears to selectively degrade the second strand under basal conditions, while phage infection correlates with enhanced first-strand accumulation. A phage-encoded protein with predicted SSB-like architecture and a conserved C-terminal tip motif is required for efficient DRT10 activation during infection. Together, these findings establish DRT10 as a surveillance system whose activation threshold is jointly controlled by constitutive cDNA synthesis, host exonuclease activity, and a phage-encoded SSB-like trigger that perturbs host ssDNA metabolism. We propose a model in which accumulation of DRT10-derived cDNA triggers activation of the SLATT transmembrane effector to initiate immune defense.

microbiology↗

bioRxiv2026

The spliceosome assembles on excised linear introns to protect them from degradation

In Saccharomyces cerevisiae, prolonged cellular stress induces some introns to accumulate post-splicing as stable, linear, spliceosome-protected RNAs1. These stable introns are defined by having short distances from their branchpoint (BP) sequences to their 3'-splice sites (3'SSs). Stable introns sequester splicing components, thereby reducing splicing activity and affecting cell growth in the stressed conditions. The mechanism by which these normally ephemeral products of pre-mRNA splicing persist cannot be explained by the current understanding of the splicing pathway, which derives primarily from studies of unstressed cells and their extracts2,3. Here, we determined the cryo-electron microscopy (cryo-EM) structure of a stable-intron complex purified from saturated-culture conditions. This structure and experimental follow-up show that a Bact-like spliceosome protects stable introns from degradation, and that the short BP-3'SS distances of stable introns render this conformation of the spliceosome resistant to remodelling by helicases. Spliceosomes can also assemble onto artificial introns that have the same sequences as authentic stable introns but do not rely on splicing for their biogenesis, which demonstrates that spliceosomes arrive at this Bact-like conformation by reassembling onto linear introns after their excision from pre-mRNAs. This reassembly activity is maintained in both stressed and unstressed cells. Thus, most yeast introns compete with pre-mRNAs for access to the splicing machinery, and budding yeast has co-opted this activity to adapt to environmental insults.

molecular biology↗

bioRxiv2025

Generative design of novel bacteriophages with genome language models

Many important biological functions arise not from single genes, but from complex interactions encoded by entire genomes. Genome language models have emerged as a promising strategy for designing biological systems, but their ability to generate functional sequences at the scale of whole genomes has remained untested. Here, we report the first generative design of viable bacteriophage genomes. We leveraged frontier genome language models, Evo 1 and Evo 2, to generate whole-genome sequences with realistic genetic architectures and desirable host tropism, using the lytic phage {Phi}X174 as our design template. Experimental testing of AI-generated genomes yielded 16 viable phages with substantial evolutionary novelty. Cryo-electron microscopy revealed that one of the generated phages utilizes an evolutionarily distant DNA packaging protein within its capsid. Multiple phages demonstrate higher fitness than {Phi}X174 in growth competitions and in their lysis kinetics. A cocktail of the generated phages rapidly overcomes {Phi}X174-resistance in three E. coli strains, demonstrating the potential utility of our approach for designing phage therapies against rapidly evolving bacterial pathogens. This work provides a blueprint for the design of diverse synthetic bacteriophages and, more broadly, lays a foundation for the generative design of useful living systems at the genome scale.

synthetic biology↗

bioRxiv2024

Control of 3' splice site selection by the yeast splicing factor Fyv6

Pre-mRNA splicing is catalyzed in two steps: 5' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (1st and 2nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2nd step factor in S. cerevisiae; however, we did not determine how widespread Fyv6s impact is on the transcriptome. To answer this question, we have used RNA-seq to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3' SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-EM structure of a yeast product complex spliceosome containing Fyv6 at 2.3 [A]. The structure reveals that Fyv6 is the only 2nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv61 suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3' SS.

biochemistry↗

bioRxiv2024

Similar mechanisms of retron-mediated anti-phage defense for different families of tailed phages

Bacterial retrons are tripartite systems consisting of a cognate non-coding RNA, reverse transcriptase, and additional (effector) protein with diverse predicted enzymatic functions. In this study, we investigated the role and mechanism of Retron-Eco11, a novel type III-A3 retron system associated with a phosphoribosyltransferase-like effector protein, in phage defense. Here, we show that the Retron-Eco11 tripartite system protects against phage infection and that UvsW and D10, two functional homolog helicases found in T4 and T5 phages, respectively, serve as specific triggers for the Retron-Eco11 defense system. Our findings confirmed that msDNA and both protein components of the intron complex are indispensable for its protective function. Once the retron system detects the activity of these helicase proteins, it activates the toxicity of the effector protein bound to the retron complex, leading to an abortive infection. These findings underscore the application of a comparable anti-phage defense strategy using Retron-Eco11 across diverse phage families. This should aid in deciphering the processes through which the Retron complex detects and identifies the invading phages.

microbiology↗

bioRxiv2023

Data-driven regularisation lowers the size barrier of cryo-EM structure determination

Macromolecular structure determination by electron cryo-microscopy (cryo-EM) is limited by the alignment of noisy images of individual particles. Because smaller particles have weaker signals, alignment errors impose size limitations on its applicability. Here, we explore how image alignment is improved by the application of deep-learning to exploit prior knowledge about biological macromolecular structures that would otherwise be difficult to express mathematically. We train a denoising convolutional neural network on pairs of half-set reconstructions from the electron microscopy data bank (EMDB) and use this denoiser as an alternative to a commonly used smoothness prior. We demonstrate that this approach, which we call Blush regularisation, yields better reconstructions than existing algorithms, in particular for data with low signal-to-noise ratios. The reconstruction of a protein-nucleic acid complex with a molecular weight of 40 kDa, which was previously intractable, illustrates that regularisation through denoising will expand the applicability of cryo-EM structure determination for a wide range of biological macromolecules.

biophysics↗

bioRxiv2020

Structural basis for conformational equilibrium of the catalytic spliceosome

The catalytic spliceosome exists in equilibrium between the branching (B*/ C) and exon ligation (C*/ P) conformations. Here we present the electron cryo-microscopy reconstruction of the Saccharomyces cerevisiae C complex at 2.8 [A] resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. In the Ci conformation, the exon-ligation factors Prp18 and Slu7 are already bound before ATP hydrolysis by Prp16, which destabilises the branching conformation. Biochemical assays suggest these pre-bound factors prime C complex for conversion to C* by Prp16. A complete model of the Prp19-complex (NTC) shows how the NTC pre-recruits the branching factors Yju2 and Isy1 before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain associated with the C* and P spliceosomes and promote exon ligation. Our results explain how Prp16 action modulates dynamic binding of step-specific factors to alternatively stabilise the C or C* conformation and establish equilibrium of the catalytic spliceosome.

biophysics↗

bioRxiv2017

Post-catalytic spliceosome structure reveals mechanism of 3'-splice site selection

Introns are removed from eukaryotic mRNA precursors by the spliceosome in two transesterification reactions - branching and exon ligation. Following branching, the 5'-exon remains paired to U5 snRNA loop 1, but the mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7[A] cryo-EM structure of the yeast P complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognised through non-Watson-Crick pairing with the 5'-splice site and the branch point adenosine. A conserved loop of Prp18 together with the -finger and the RNaseH domain of Prp8 clamp the docked 3'-splice site and 3'-exon. The step 2 factors Prp18 and Slu7 and the C-terminal domain of Yju2 stabilise a conformation competent for 3'-splice site docking and exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides of the introns and provides insight into the catalytic mechanism of exon ligation.

molecular biology↗