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Williams, L. D.

Publications and source records attributed to Williams, L. D..

At least 19 recordsLinked to original sources

Deep shifts in Evolutionary Rate Trajectories of Ancient Bacterial Genes

Reconstruction of ancestral gene repertoires from extant genomes captures only the genes that survived; those lost from the record are invisible. Among the genes that did persist, selective pressures change not only across lineages but across time. Here, we resolve evolutionary rate trajectories across 528 genes in the Last Bacterial Common Ancestor (LBCA). We hypothesized that LBCA genes would show distinct evolutionary rate trajectories across bacterial history and tested this by resolving normalized branch lengths across five calibrated taxonomic intervals (phylum, class, order, family, and genus), spanning approximately 2.1 billion years of bacterial diversification. The distribution of rate trajectories is continuous, but four clusters capture the major patterns: Decelerating, Class-Peaking, Constant, and Accelerating. The Decelerating and Class-Peaking clusters are composed predominantly of Genetic Information Processing genes, whereas the Constant and Accelerating clusters are enriched for Metabolic genes. Specifically, the Decelerating cluster is enriched for core informational machinery, including translation initiation factors and components of the expressome, the molecular complex physically coupling transcription and translation, suggesting that transcription-translation interfaces locked in early in bacterial history. Cofactor-dependence and cofactor biosynthesis are decoupled: biosynthetic pathways producing metallocofactors such as heme, molybdopterin, and cobalamin, concentrated in the Constant and Accelerating clusters but proportionally more proteins use inorganic cofactors in the Decelerating and Class-Peaking clusters. This offset coincides with the shift in metal bioavailability associated with the Great Oxidation Event and reveals genomic fingerprints of the co-evolution of bacterial metabolism with planetary geochemistry.

evolutionary biology↗

Ex vivo recapitulation of intramuscular mRNA vaccination with naive and recall antigens using a human Lymphoid Follicle Chip platform

Predicting the efficacy and toxicity of intramuscular mRNA vaccines remains challenging. Here, we describe an ex vivo human cell-based model that replicates immune responses to lipid nanoparticle (LNP)-based mRNA vaccines that require intramuscular injection. Vaccines are administered into a biomimetic muscle module containing human skeletal myoblasts and antigen-presenting cells (APCs) to mimic intramuscular vaccination, followed by transfer of the APCs and soluble factors to a microfluidic human lymphoid follicle chip (LF Chip) to mimic lymphatic drainage. Non-replicating mRNA vaccines directly induce antigen expression in APCs, whereas self-amplifying mRNA vaccines require muscle cell-APC contact within the intramuscular vaccination module. Transfer of APCs and soluble factors to the LF Chip induces LF expansion, de novo antigen-specific IgG production against a naive antigen (rabies virus glycoprotein), and cytokine release, with responses varying depending on LNP type. Vaccination of LF chips against SARS- COV-2 Spike recall antigen using the Moderna Spikevax vaccine generates neutralizing antibodies and induces somatic hypermutation. This biomimetic platform offers an all-human alternative for evaluating vaccine-induced immunity, potentially obviating the need for non-human primates and accelerating vaccine development.

immunology↗

R2DT: a comprehensive platform for visualising RNA secondary structure

RNA secondary (2D) structure visualisation is an essential tool for understanding RNA function. R2DT is a software package designed to visualise RNA 2D structures in consistent, recognisable, and reproducible layouts. The latest release, R2DT 2.0, introduces multiple significant features, including the ability to display position-specific information, such as single nucleotide polymorphisms (SNPs) or SHAPE reactivities. It also offers a new template-free mode allowing visualisation of RNAs without pre-existing templates, alongside a constrained folding mode and support for animated visualisations. Users can interactively modify R2DT diagrams, either manually or using natural language prompts, to generate new templates or create publication-quality images. Additionally, R2DT features faster performance, an expanded template library, and a growing collection of compatible tools and utilities. Already integrated into multiple biological databases, R2DT has evolved into a comprehensive platform for RNA 2D visualisation, accessible at https://r2dt.bio.

bioinformatics↗

BEAN and HABAS: Polyphyletic insertions in RNAP that point to deep evolutionary divergence of bacteria

The {beta} and {beta} subunits of the RNA polymerase (RNAP) are large proteins with complex multi-domain architectures that include several insertional domains. Here, we analyze the multi-domain organizations of bacterial RNAP-{beta} and RNAP-{beta} using sequence, experimentally determined structures and AlphaFold structure predictions. We observe that bacterial lineage-specific domains in RNAP-{beta} belong to a group of domains that we call BEAN (Broadly Embedded ANnex) and that in RNAP-{beta}, bacterial lineage-specific domains are HAmmerhead/BArrel-Sandwich Hybrid (HABAS) domains. The BEAN domain has a characteristic three-dimensional structure composed of two square bracket-like elements that are antiparallel relative to each other. The HABAS domain contains a four-stranded open {beta}-sheet with a GD-box-like motif in one of the {beta}-strands and the adjoining loop. The BEAN domain is identified not only in the bacterial RNAP-{beta}, but also in the archaeal version of universal ribosomal protein L10. The HABAS domain is observed as an insertional domain in several metabolic proteins. The phylogenetic distributions of bacterial lineage-specific insertional domains of {beta} and {beta} subunits of RNAP follow the Tree of Life. The presence of insertional domains can help establish a relative timeline of events in the evolution of a protein because insertion is inferred to post-date the base domain. We discuss mechanisms that might account for the discovery of homologous insertional domains in non-equivalent locations in bacteria and archaea.

evolutionary biology↗

Tailoring Tfh Profiles Enhances Antibody Persistence to a Clade C HIV-1 Vaccine in Rhesus Macaques

CD4 T follicular helper cells (Tfh) are essential for establishing serological memory and have distinct helper attributes that impact both the quantity and quality of the antibody response. Insights into Tfh subsets that promote antibody persistence and functional capacity can critically inform vaccine design. Based on the Tfh profiles evoked by the live attenuated measles virus vaccine, renowned for its ability to establish durable humoral immunity, we investigated the potential of a Tfh1/17 recall response during the boost phase to enhance persistence of HIV-1 Envelope (Env) antibodies in rhesus macaques. Using a DNA-prime encoding gp160 antigen and Tfh polarizing cytokines (interferon protein-10 (IP-10) and interleukin-6 (IL-6)), followed by a gp140 protein boost formulated in a cationic liposome-based adjuvant (CAF01), we successfully generated germinal center (GC) Tfh1/17 cells. In contrast, a similar DNA-prime (including IP-10) followed by gp140 formulated with monophosphoryl lipid A (MPLA)+QS-21 adjuvant predominantly induced GC Tfh1 cells. While the generation of GC Tfh1/17 cells with CAF01 and GC Tfh1 cells with MPLA+QS-21 induced comparable peak Env antibodies, the latter group demonstrated significantly greater antibody concentrations at week 8 after final immunization which persisted up to 30 weeks (gp140 IgG ng/ml-MPLA; 5500; CAF01, 2155; p <0.05). Notably, interferon {gamma}+ Env-specific Tfh responses were consistently higher with gp140 in MPLA+QS-21 and positively correlated with Env antibody persistence. These findings suggest that vaccine platforms maximizing GC Tfh1 induction promote persistent Env antibodies, important for protective immunity against HIV.

immunology↗

Common evolutionary origins of the bacterial glycyl tRNA synthetaseand alanyl tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) establish the genetic code. Each aaRS covalently links a given canonical amino acid to a cognate set of tRNA isoacceptors. Glycyl tRNA aminoacylation is unusual in that it is catalyzed by different aaRSs in different lineages of the Tree of Life. We have investigated the phylogenetic distribution and evolutionary history of bacterial glycyl tRNA synthetase (bacGlyRS). This enzyme is found in early diverging bacterial phyla such as Firmicutes, Acidobacteria, and Proteobacteria, but not in archaea or eukarya. We observe relationships between each of six domains of bacGlyRS and six domains of four different RNA-modifying proteins. Component domains of bacGlyRS show common ancestry with i) the catalytic domain of class II tRNA synthetases; ii) the HD domain of the bacterial RNase Y; iii) the body and tail domains of the archaeal CCA-adding enzyme; iv) the anti-codon binding domain of the arginyl tRNA synthetase; and v) a previously unrecognized domain that we call ATL (Ancient tRNA latch). The ATL domain is found only in bacGlyRS and in the universal alanyl tRNA synthetase (uniAlaRS). Further, the catalytic domain of bacGlyRS is more closely related to the catalytic domain of uniAlaRS than to any other aminoacyl tRNA synthetase. The combined data suggest that the ATL and catalytic domains of these two enzymes are ancestral to bacGlyRS and uniAlaRS, which emerged from common protein ancestors by bricolage, stepwise accumulation of protein domains, before the last universal common ancestor of life.

evolutionary biology↗

Translation in a Box: Orthogonal Evolution in the Saccharomyces cerevisiae Mitochondrion

The ability to re-engineer and creatively evolve the translation system (TS) would allow invention of new coded polymers by altering the amino acid sidechain inventory and by shifting the polypeptide backbone into new chemical spaces. Unfortunately, the TS is difficult to manipulate and is more constrained over evolution than any other biological system. An orthogonal TS, running in parallel to the primary TS within a given host cell, would release constraints and allow TS manipulation. A fully orthogonal TS requires dedicated rRNAs, rProteins, aminoacyl-tRNA synthetases, and initiation and termination factors, none of which interact with the primary TS. The S. cerevisiae mitochondrial TS is fully orthogonal to the cytosolic TS. Mito-rRNAs, mito- rProteins, mito-tRNAs, mito-aminoacyl tRNA synthetases, and mito-translation factors are distinct from, physically separated from, and functionally independent of their cytosolic counterparts. Here, the S. cerevisiae mitochondrial translation system was subjected to various stresses including antibiotics, mutagenesis and truncation of mito-rProteins, or wholesale replacement of mito-rProteins. Directed evolution of these stressed systems was facilitated by controlled transitions between fermentation and respiration, by changing the carbon source in the growth medium; the dependence of S. cerevisiae survival on mitochondrial translation can be toggled on and off. Specific recreation of the resulting mutations recapitulate the evolved phenotypes. The method developed here appears to be a general approach for discovering functional dependencies. Suppressor mutations reveal functional dependencies within the S. cerevisiae mitochondrial TS. For example proteins Rrg9 or Mrx1 interact with the mito-TS and have critical role in its function. The combined results indicate that the S. cerevisiae mitochondrial TS can be engineered and evolved in isolation of the cytosolic TS. SignificanceThe Central Dogma of Molecular Biology rules life on Earth. Information flows from DNA to mRNA to protein. In the last step of the Central Dogma, the translation system decodes mRNA and produces coded proteins by linking amino acids into polymers. Engineering and evolving the translation system could permits full technical control over this process and could lead to the generation of novel polymers. Here, we use the mitochondrial translation system in the budding yeast Saccharomyces cerevisiae for directed evolution of translation. We modify and evolve the translation system both directly and indirectly using antibiotics and gene editing tools and then measure resulting functionality. Our results show this secondary translation system inside S. cerevisiae mitochondria can be used as an approach for translation engineering.

synthetic biology↗

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin and finally elution and identification by mass spectrometry. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in a SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization and vesicular trafficking, many of which have been associated with heme through complimentary studies published recently. Taken together, these results provide support for the emerging role for heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

biochemistry↗

Fold Evolution by Creative Destruction

Mechanisms by which new protein folds emerge and diverge pose central questions in biological sciences. Incremental mutation and step-wise adaptation explain relationships between topologically similar protein folds. However, the universe of folds is diverse and riotous, suggesting roles of more potent and creative forces. Sequence and structure similarity are observed between topologically distinct folds, indicating that proteins with distinct folds may share common ancestry. We found evidence of common ancestry between three distinct {beta}-barrel folds: OB, SH3 and cradle loop barrel (CLB). The data suggest a mechanism of fold evolution that interconverts SH3, OB and CLB. This mechanism, which we call creative destruction, can be generalized to explain other examples of fold evolution including circular permutation. In creative destruction, an open reading frame duplicates or otherwise merges with another to produce a fused polypeptide. A merger forces two ancestral domains into a new sequence and spatial context. The fused polypeptide can explore folding landscapes that are inaccessible to either of the independent ancestral domains. However, the folding landscapes of the fused polypeptide are not fully independent of those of the ancestral domains. Creative destruction is thus partially conservative in that a daughter fold would inherit some motifs from the ancestral folds. After a merger and refolding, adaptive processes such as mutation and loss of extraneous segments optimize the new daughter fold. SignificanceMechanisms of emergence and early diversification of structured proteins present deep and difficult problems in evolutionary biology. Here we excavate the deepest evolutionary history, found within the translation machinery, which is an ancient molecular fossil and the birthplace of all proteins. We demonstrate common origins of some of the simplest, oldest and most common protein folds. Furthermore, the data suggest a mechanism, that we call creative destruction, that explains at molecular level how simple folds spawn more complex folds. In this mechanism, new folds emerge from old folds via gene duplication, expression, exploration of new folding landscapes and adaptation. Creative destruction explains the facile emergence of complex from simple architectures in a funneled exploration.

evolutionary biology↗

RNA and the Goldilocks Zone: Where Mg2+ concentration is just right

Magnesium, the most abundant divalent cation in cells, catalyzes RNA cleavage but also promotes RNA folding. Because folding can protect RNA from cleavage, we predicted a "Goldilocks peak", which is a local maximum in RNA lifetime at the Mg2+ concentration required for folding. Here we use simulation and experiment to discover an innate yet sophisticated mechanism of control of RNA lifetime. By simulation we characterized the RNA Goldilocks peak and its dependence on cleavage parameters and extent of folding. Supporting experiments with yeast tRNAPhe and Tetrahymena ribozyme P4-P6 domains show that structured RNA can inhabit a Goldilocks peak in vitro. The Goldilocks peaks are tunable by differences in cleavage rate constants, Mg2+ binding cooperativity, and Mg2+ affinity. Broad ranges of those folding and cleavage parameters produce Goldilocks peaks of different intensities. Goldilocks behavior allows ultrafine control of RNA chemical lifetime, whereas non-folding RNAs do not display a Goldilocks peak. In sum, the effects of Mg2+ on RNA persistence are expected to be pleomorphic, both protecting and degrading RNA. In evolutionary context, Goldilocks behavior may have shaped RNA in an early Earth environment containing Mg2+ and other metals.

biochemistry↗

Monocyte-derived transcriptome signature indicates antibody-dependent cellular 1 phagocytosis as the primary mechanism of vaccine-induced protection against HIV-1

A gene signature previously correlated with mosaic adenovirus 26 vaccine protection in simian immunodeficiency virus (SIV) and SHIV challenge models in non-human primates (NHP). In this report we investigated presence of this signature as a correlate of reduced risk in human clinical trials and potential mechanism for protection. The absence of this gene signature in the DNA/rAd5 human vaccine trial which did not show efficacy, strengthens our hypothesis that this signature is only enriched in studies that demonstrated protection. This gene signature was enriched in the partially effective RV144 human trial that administered the ALVAC/protein vaccine, and we find that the signature associates with both decreased risk of HIV-1 acquisition and increased vaccine efficacy. Total RNA-seq in a clinical trial that used the same vaccine regimen as the RV144 HIV vaccine implicated antibody-dependent cellular phagocytosis (ADCP) as a potential mechanism of vaccine protection. CITE-seq profiling of 53 surface markers and transcriptomes of 53,777 single cells from the same trial, showed that genes in this signature were primarily expressed in cells belonging to the myeloid lineage including monocytes, which are major effector cells for ADCP. The consistent association of this transcriptome signature with vaccine efficacy represents a tool both to identify potential mechanisms, as with ADCP here, and to screen novel approaches to accelerate development of new vaccine candidates.

systems biology↗

Is Palmer's elm leaf goldenrod real? The Angiosperms353 kit provides within-species signal in Solidago ulmifolia s.l.

The genus Solidago represents a taxonomically challenging group due to its sheer number of species, putative hybridization, polyploidy, and shallow genetic divergence among species. Here we use a dataset obtained exclusively from herbarium specimens to evaluate the status of Solidago ulmifolia var. palmeri, a morphologically subtle taxon potentially confined to Alabama, Arkansas, Mississippi, and Missouri. A multivariate analysis of both discrete and continuous morphological data revealed no clear distinction between S. ulmifolia var. palmeri and Solidago ulmifolia var. ulmifolia. Solidago ulmifolia var. palmeris status was also assessed with a phylogenomic and SNP clustering analysis of data generated with the "Angiosperms353" probe kit. Neither analysis supported Solidago ulmifolia var. palmeri as a distinct taxon, and we suggest that this name should be discarded. The status of Solidago delicatula (formerly known as Solidago ulmifolia var. microphylla) was also assessed. Both morphological and phylogenic analyses supported the species status of S. delicatula and we suggest maintaining this species at its current rank. These results highlight the utility of the Angiosperms353 probe kit, both with herbarium tissue and at lower taxonomic levels. Indeed, this is the first study to utilize this kit to identify genetic groups within a species.

evolutionary biology↗

On the potential of Angiosperms353 for population genomics.

Targeted sequencing using Angiosperms353 has emerged as a low-cost tool for phylogenetics, with early results spanning scales from all flowering plants to within genera. The use of universal markers at narrower scales--within populations-- would eliminate the need for specific marker development while retaining the benefits of full-gene sequences. However, it is unclear whether the Angiosperms353 markers provide sufficient variation within species to calculate demographic parameters. Using herbarium specimens from a 50-year-old floristic survey of Guadalupe Mountains National Park, we sequenced 95 samples from 24 species using Angiosperms353. We adapted a data workflow to process targeted sequencing data that calls variants within each species and prepares data for population genetic analysis. We calculated genetic diversity using standard metrics (e.g. heterozygosity, Tajimas D). Angiosperms353 gene recovery was associated with genomic library concentration, with limited phylogenetic bias. We identified over 1000 segregating variants with zero missing data within 22 of 24 species. A subset of these variants, which were filtered to remove linked SNPs, revealed high heterozygosity in many species. Tajimas D calculated within each species indicated a moderate number of markers potentially under selection and identified evidence of population bottlenecks in some species. Despite sequencing few individuals per species, the Angiosperms353 markers contained sufficient variation calculate demographic parameters. Larger sampling within species will allow for estimating gene flow and population dynamics in any angiosperm. Our study will benefit conservation genetics, where Angiosperms353 provides universal repeatable markers, low missing data, and haplotype information.

evolutionary biology↗

R2DT: computational framework for template-based RNA secondary structure visualisation across non-coding RNA types

Non-coding RNAs (ncRNA) are essential for all life, and the functions of many ncRNAs depend on their secondary (2D) and tertiary (3D) structure. Despite proliferation of 2D visualisation software, there is a lack of methods for automatically generating 2D representations in consistent, reproducible, and recognisable layouts, making them difficult to construct, compare and analyse. Here we present R2DT, a comprehensive method for visualising a wide range of RNA structures in standardised layouts. R2DT is based on a library of 3,632 templates representing the majority of known structured RNAs, from small RNAs to the large subunit ribosomal RNA. R2DT has been applied to ncRNA sequences from the RNAcentral database and produced >13 million diagrams, creating the worlds largest RNA 2D structure dataset. The software is freely available at https://github.com/rnacentral/R2DT and a web server is found at https://rnacentral.org/r2dt.

bioinformatics↗

Human Ribosomal G-Quadruplexes Regulate Heme Bioavailability

The in vitro formation of stable G-quadruplexes (G4s) in human ribosomal RNA (rRNA) was recently reported. However, their formation in cells and their cellular roles have not been resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest ribosomes are central hubs of heme metabolism.

biochemistry↗

Eukaryotic-like ribosomal RNA region in Lokiarchaeota

The ribosomes common core, comprised of ribosomal RNA (rRNA) and universal ribosomal proteins, connects all life back to a common ancestor and serves as a window to relationships among organisms. The rRNA of the common core is most similar to rRNA of extant bacteria. In eukaryotes, the rRNA of the common core is decorated by expansion segments (ESs) that vastly increase its size. Supersized ESs have not been observed previously in Archaea, and the origin of eukaryotic ESs remains enigmatic. We discovered that the large subunit (LSU) rRNA of two Asgard phyla, Lokiarchaeota and Heimdallarchaeota, considered to be the closest modern archaeal cell lineages to Eukarya, bridge the gap in size between prokaryotic and eukaryotic LSU rRNA. The elongated LSU rRNAs in Lokiarchaeota and Heimdallarchaeota stem from the presence of two supersized ESs, ES9 and ES39. We applied chemical footprinting experiments to study the structure of Lokiarchaeota ES39. Furthermore, we used covariation and sequence analysis to study the evolution of Asgard ES39s and ES9s. By defining the common eukaryotic ES39 signature fold, we found that Asgard ES39s have more and longer helices than eukaryotic ES39s. While Asgard ES39s have sequences and structures distinct from eukaryotic ES39s, we found overall conservation of a three-way junction across the Asgard species that matches eukaryotic ES39 topology, a result consistent with the accretion model of ribosomal evolution. SignificanceEukaryotes possess longer and more complex ribosomal RNA (rRNA) than Bacteria and Archaea, including eukaryotic-specific rRNA expansion segments (ESs). The origin and evolution of ESs has long remained a mystery. We show that two recently discovered Asgard archaeal phyla, Lokiarchaeota and Heimdallarchaeota, contain large subunit rRNA with ESs are "supersized" (>100 nt) compared to all other prokaryotes studied to date. Asgard ESs have distinct structures from eukaryotic ESs, but share a common three-way junction out of which the ES grew. Our findings raise the possibility that supersized ESs existed on the ribosomal surface before the last eukaryotic common ancestor, opening the question of whether ribosomal complexity is more deeply rooted than previously thought.

evolutionary biology↗

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosomes early evolution in a high Fe2+ environment, and its continued use by obligate anaerobes inhabiting high Fe2+ niches. Here we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order rate constant with respect to divalent cations is more than 200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+, and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because it is tightly coordinated by rRNA and buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity. Key PointsO_LIFe2+ cleaves rRNA by a non-oxidative in-line cleavage mechanism that is more than 200 times faster than in-line cleavage with Mg2+; C_LIO_LIribosomes purified from cells grown under low O2 and high Fe2+ retain ~10 Fe2+ ions per ribosome and produce as much protein as low O2, high Mg2+-grown ribosomes; C_LIO_LIa small fraction (~2%) of Fe2+ that is associated with the ribosome is not exchangeable. C_LI

molecular biology↗

Dynamic G-quadruplexes on the surface of the human ribosome

Profound similarities and critical differences mark ribosomes across phylogeny. The ribosomal core, approximated by the prokaryotic ribosome, is universal, yet mammalian ribosomes are nearly twice as large as those of prokaryotes. Differences in size are due in part to rRNA expansion segments. Here we show rRNA tentacles of Expansion Segment 7 (ES7) of Homo sapiens can form G-quadruplexes in vitro. G-quadruplex-forming regions are located on the most surface-exposed regions of the ribosome, near the termini of rRNA tentacles. We characterized rRNA of the large ribosomal subunit by computation, circular dichroism, gel mobility, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts and blotting. We investigated ES7 and oligomers derived from ES7, intact 28S rRNA, and 80S ribosomes and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. Proteins that associate with rRNA G-quadruplexes include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins (hnRNPs). And finally, by multiple sequence alignments, we observed that G-quadruplex-forming sequences appear to be a general feature LSU rRNA of the phylum Chordata but not in other phyla. It is known that G-quadruplexes form in telomeres, promoters, and untranslated regions of mRNA but, to our knowledge, they have not been reported previously in ribosomes.

biochemistry↗