Biology subjects

Webster, M. W.

Publications and source records attributed to Webster, M. W..

7 recordsLinked to original sources

bioRxiv2026

Conservation and evolution of the ribosome across the photosynthetic lineage

Regulated biogenesis of photosynthetic proteins by the chloroplast ribosomes is central to plant development and environmental adaptation. Here, we describe changes to the ribosome that occurred during evolution of chloroplasts from cyanobacteria in the photosynthetic lineage based on structural, proteomic and bioinformatic analyses. We identify structural features common to oxygenic photosynthetic organisms and distinct from other bacteria, as well as the gain and loss of ribosomal proteins, rRNA modifications and hibernation factors upon endosymbiosis. We uncover structural variation among land plant lineages at sites that remodel the peptide exit tunnel and features associated with translation initiation. The data challenge the prevailing view of ribosomal features that are specific to the chloroplast by demonstrating that many originated earlier, in cyanobacteria, or later, during land plant evolution.

biochemistry↗

bioRxiv2025

Stepwise and lineage-specific divergence of a major immune co-chaperone complex in leptosporangiate ferns

Protein-protein interactions are essential for proper cellular function and are often under strong evolutionary pressures to maintain their stability and specificity. In plants, a broadly distributed chaperone complex comprised of the RAR1 (REQUIRED FOR MLA12 RESISTANCE 1) and SGT1 (SUPRESSOR OF THE G2 ALLELE OF SKP1) co-chaperones alongside the HSP90 (HEAT SHOCK PROTEIN90) core chaperone are important for immunity. Despite its importance in flowering plants, a deeper understanding of how this complex evolved remains limited. Here, we examine the molecular evolutionary history of the RAR1-SGT1 interaction across land plants. We identified a lineage-specific divergence of the RAR1-SGT1 binding interface in vascular non-seed ferns, which renders orthologs unable to interact outside of their lineage. Further investigation of interface diversity uncovered a single amino acid residue in RAR1 and three corresponding residues in SGT1 that collectively dictate binding specificity. Ancestral state reconstruction supported stepwise evolution of specificity in SGT1 in leptopsporangiate ferns, which was initiated by a promiscuous intermediate state that widened its capacity to bind RAR1 before subsequent mutations locked in specificity. Our data highlight the broad conservation of the RAR1-SGT1 interface and the coevolutionary dynamics that shaped interface maintenance during lineage-specific diversification.

plant biology↗

bioRxiv2025

Structural analysis of the NifL-NifA complex reveals the molecular basis of anti-activation of nitrogen fixation gene expression in Azotobacter vinelandii

Understanding the molecular basis of regulated nitrogen (N2) fixation is essential for engineering N2-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system NifL-NifA controls the expression of nif genes that encode the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. Whilst genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-EM map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.

microbiology↗

bioRxiv2024

A disease resistance protein triggers oligomerization of its NLR helper into a hexameric resistosome to mediate innate immunity

NRCs are essential helper NLR (nucleotide-binding domain and leucine-rich repeat) proteins that execute the immune response triggered by disease resistance proteins, also known as sensor NLRs. The structure of the resting state of NbNRC2 was recently revealed to be a homodimer. However, the sensor-activated state has not yet been elucidated. In this study, we used cryo-EM to determine the structure of sensor-activated NbNRC2, which forms a hexameric inflammasome-like structure known as resistosome. To confirm the functional significance of the hexamer, we mutagenized the interfaces involved in oligomerization and found that mutations in three nucleotide-binding domain interface residues abolish oligomerization and immune signalling. Comparative structural analyses between the resting state NbNRC2 homodimer and the sensor-activated homohexamer revealed significant structural rearrangements before and after activation, providing insights into NLR activation mechanisms. Furthermore, structural comparisons between the NbNRC2 hexamer and previously reported CC-NLR pentameric assemblies revealed features in NbNRC2 that allow for the integration of an additional protomer. We also used the NbNRC2 hexamer structure to assess the recently released AlphaFold 3 for the prediction of activated CC-NLR oligomers. This revealed that AlphaFold 3 allows for high-confidence modelling of the N-terminal 1-helices of NbNRC2 and other CC-NLRs, a region that has proven difficult to fully resolve using structural approaches. Overall, our work sheds light on the structural and biochemical mechanisms underpinning NLR activation and expands our understanding of NLR structural diversity.

plant biology↗

bioRxiv2024

Molecular basis of mRNA delivery to the bacterial ribosome

Protein synthesis begins with the formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through base pairing with the Shine Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S subunit. Here we examined ribosome recruitment to nascent mRNAs using cryo-EM, single-molecule fluorescence co-localization, and in-cell crosslinking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S subunit activation. Additionally, bS1 mediates the stimulation of translation initiation by RNAP. Together, our work provides a mechanistic framework for how the SD duplex, ribosomal proteins and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.

biochemistry↗

bioRxiv2020

Structural basis of transcription-translation coupling and collision in bacteria.

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The pioneering ribosome potentially contacts RNA polymerase (RNAP), forming a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here we present a series of structures representing uncoupled, coupled and collided expressome states determined by electron cryomicroscopy. A bridge between the ribosome and RNAP can be formed by the transcription factor NusG, stabilizing an otherwise variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance-channel to the RNAP exit-channel. In this collided complex, NusG-linkage is no longer possible. These structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study. One Sentence SummaryStructures of the molecular assembly executing gene expression shed light on transcription translation coupling.

biochemistry↗

bioRxiv2018

The mechanism of Ccr4-Not recruitment to specific mRNAs involves sequence-selective tethering by RNA-binding proteins

The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs). We find that the fission yeast homologues of Tristetraprolin/TTP and Pumilio/Puf (Zfs1 and Puf3) act as molecular tethers: They recruit Ccr4-Not via multiple regions within low-complexity sequences, and bind specific RNA sequences via RNA-binding domains. Using a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs, we demonstrate that Puf3 can distinguish between RNAs of very similar sequence. This is primarily due to differences in the dissociation rate constants. As a result, motif quality is a major determinant of mRNA stability for Puf3 targets in vivo. Together, we provide new insight into the selective deadenylation of specific mRNAs by Ccr4-Not, and the prediction of targeted mRNAs.

molecular biology↗