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Eric Westhof

Publications and source records attributed to Eric Westhof.

At least 19 recordsLinked to original sources
PubMed2026

Phosphate backbone epitranscriptomics: Discovery of natural RNA phosphorothioates and their writer machinery.

Over 150 modifications expand the RNA alphabet, yet all known natural modifications occur on nucleobases or ribose sugars, with none identified on the phosphate backbone. In contrast, phosphorothioates (PSs), in which a non-bridging phosphate oxygen is replaced with sulfur, are central to RNA therapeutics but have never been reliably detected in natural RNAs. Here, we develop sequencing- and mass spectrometry-based approaches to quantitatively map RNA PSs at single-nucleotide resolution. Across diverse archaeal species, we identify stereospecific PS modifications at rRNA and tRNA hotspots, which are dynamically regulated by sulfur availability and temperature. We uncover a diverse enzyme family that selectively modifies tRNA/rRNA substrates and whose evolutionary presence/absence matches the distribution of PSs. Enzyme loss causes inviability or temperature sensitivity, and functional analyses reveal that tRNA PSs enhance tRNA stability. These findings establish the first natural RNA phosphate-backbone modification and its enzymatic machinery, providing a foundation for mechanistic and functional exploration.

RNA modifications↗
PubMed2006

Structure of the ribosome-bound cricket paralysis virus IRES RNA.

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.

Animals↗
PubMed2006

Crystal structure of the bacterial ribosomal decoding site complexed with amikacin containing the gamma-amino-alpha-hydroxybutyryl (haba) group.

Amikacin is the 4,6-linked aminoglycoside modified at position N1 of the 2-deoxystreptamine ring (ring II) by the L-haba group. In the present study, the crystal structure of a complex between oligonucleotide containing the bacterial ribosomal A site and amikacin has been solved at 2.7 A resolution. Amikacin specifically binds to the A site in practically the same way as its parent compound kanamycin. In addition, the L-haba group interacts with the upper side of the A site through two direct contacts, O2*...H-N4(C1496) and N4*-H...O6(G1497). The present crystal structure shows how the introduction of the L-haba group on ring II of aminoglycoside is an effective mutation for obtaining a higher affinity to the bacterial A site.

Amikacin↗
PubMed2006

The building blocks and motifs of RNA architecture.

RNA motifs can be defined broadly as recurrent structural elements containing multiple intramolecular RNA-RNA interactions, as observed in atomic-resolution RNA structures. They constitute the modular building blocks of RNA architecture, which is organized hierarchically. Recent work has focused on analyzing RNA backbone conformations to identify, define and search for new instances of recurrent motifs in X-ray structures. One current view asserts that recurrent RNA strand segments with characteristic backbone configurations qualify as independent motifs. Other considerations indicate that, to characterize modular motifs, one must take into account the larger structural context of such strand segments. This follows the biologically relevant motivation, which is to identify RNA structural characteristics that are subject to sequence constraints and that thus relate RNA architectures to sequences.

Base Pairing↗
PubMed2006

Proximity of conserved U6 and U2 snRNA elements to the 5' splice site region in activated spliceosomes.

Major structural changes occur in the spliceosome during its catalytic activation, which immediately precedes the splicing of pre-mRNA. Whereas changes in snRNA conformation are well documented at the level of secondary RNA-RNA interactions, little is known about the tertiary structure of this RNA-RNA network, which comprises the spliceosome's catalytic core. Here, we have used the hydroxyl-radical probe Fe-BABE, tethered to the tenth nucleotide (U(+10)) of the 5' end of a pre-mRNA intron, to map RNA-RNA proximities in spliceosomes. These studies revealed that several conserved snRNA regions are close to U(+10) in activated spliceosomes, namely (i) the U6 snRNA ACAGAG-box region, (ii) portions of the U6 intramolecular stem-loop (U6-ISL) including a nucleotide implicated in the first catalytic step (U74), and (iii) the region of U2 that interacts with the branch point. These data constrain the relative orientation of these structural elements with respect to U(+10) in the activated spliceosome. Upon conversion of the activated spliceosome to complex C, the accessibility of U6-ISL to hydroxyl-radical cleavage is altered, suggesting rearrangements after the first catalytic step.

Base Sequence↗
PubMed2006

The RNA polymerase III-dependent family of genes in hemiascomycetes: comparative RNomics, decoding strategies, transcription and evolutionary implications.

We present the first comprehensive analysis of RNA polymerase III (Pol III) transcribed genes in ten yeast genomes. This set includes all tRNA genes (tDNA) and genes coding for SNR6 (U6), SNR52, SCR1 and RPR1 RNA in the nine hemiascomycetes Saccharomyces cerevisiae, Saccharomyces castellii, Candida glabrata, Kluyveromyces waltii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and the archiascomycete Schizosaccharomyces pombe. We systematically analysed sequence specificities of tRNA genes, polymorphism, variability of introns, gene redundancy and gene clustering. Analysis of decoding strategies showed that yeasts close to S.cerevisiae use bacterial decoding rules to read the Leu CUN and Arg CGN codons, in contrast to all other known Eukaryotes. In D.hansenii and C.albicans, we identified a novel tDNA-Leu (AAG), reading the Leu CUU/CUC/CUA codons with an unusual G at position 32. A systematic 'p-distance tree' using the 60 variable positions of the tRNA molecule revealed that most tDNAs cluster into amino acid-specific sub-trees, suggesting that, within hemiascomycetes, orthologous tDNAs are more closely related than paralogs. We finally determined the bipartite A- and B-box sequences recognized by TFIIIC. These minimal sequences are nearly conserved throughout hemiascomycetes and were satisfactorily retrieved at appropriate locations in other Pol III genes.

Ascomycota↗
PubMed2006

The RNA Ontology Consortium: an open invitation to the RNA community.

The aim of the RNA Ontology Consortium (ROC) is to create an integrated conceptual framework-an RNA Ontology (RO)-with a common, dynamic, controlled, and structured vocabulary to describe and characterize RNA sequences, secondary structures, three-dimensional structures, and dynamics pertaining to RNA function. The RO should produce tools for clear communication about RNA structure and function for multiple uses, including the integration of RNA electronic resources into the Semantic Web. These tools should allow the accurate description in computer-interpretable form of the coupling between RNA architecture, function, and evolution. The purposes for creating the RO are, therefore, (1) to integrate sequence and structural databases; (2) to allow different computational tools to interoperate; (3) to create powerful software tools that bring advanced computational methods to the bench scientist; and (4) to facilitate precise searches for all relevant information pertaining to RNA. For example, one initial objective of the ROC is to define, identify, and classify RNA structural motifs described in the literature or appearing in databases and to agree on a computer-interpretable definition for each of these motifs. To achieve these aims, the ROC will foster communication and promote collaboration among RNA scientists by coordinating frequent face-to-face workshops to discuss, debate, and resolve difficult conceptual issues. These meeting opportunities will create new directions at various levels of RNA research. The ROC will work closely with the PDB/NDB structural databases and the Gene, Sequence, and Open Biomedical Ontology Consortia to integrate the RO with existing biological ontologies to extend existing content while maintaining interoperability.

Databases, Genetic↗
PubMed2006

Docking of aminoglycosides to hydrated and flexible RNA.

Although much effort has been devoted to the development of programs suited for the docking of ligands to proteins, much less progress has been achieved in the nucleic acid field. We have developed a unique approach for docking aminoglycosides to RNA considering the flexibility of these macromolecules using conformational ensembles and accounting for the role of the first hydration shell. This concept, successfully implemented in AutoDock, relies on the computation of the intermolecular interaction energy that accounts for the presence of dynamically bound water molecules to the RNA. As an application, a set of 11 aminoglycosides was docked with an average root-mean-square deviation (RMSD) of 1.41 A to be compared with an average RMSD of 3.25 A when the original AutoDock protocol was used.

Aminoglycosides↗
PubMed2006

Two conformational states in the crystal structure of the Homo sapiens cytoplasmic ribosomal decoding A site.

The decoding A site of the small ribosomal subunit is an RNA molecular switch, which monitors codon-anticodon interactions to guarantee translation fidelity. We have solved the crystal structure of an RNA fragment containing two Homo sapiens cytoplasmic A sites. Each of the two A sites presents a different conformational state. In one state, adenines A1492 and A1493 are fully bulged-out with C1409 forming a wobble-like pair to A1491. In the second state, adenines A1492 and A1493 form non-Watson-Crick pairs with C1409 and G1408, respectively while A1491 bulges out. The first state of the eukaryotic A site is, thus, basically the same as in the bacterial A site with bulging A1492 and A1493. It is the state used for recognition of the codon/anticodon complex. On the contrary, the second state of the H.sapiens cytoplasmic A site is drastically different from any of those observed for the bacterial A site without bulging A1492 and A1493.

Animals↗
PubMed2006

Molecular contacts between antibiotics and the 30S ribosomal particle.

Crystal structures of complexes between ribosomal particles and antibiotics have pinned down very precisely the discrete binding sites of several classes of antibiotics inhibiting protein synthesis. The crystal structures of complexes between various antibiotics and ribosomal particles show definitively that ribosomal RNAs (rRNAs), rather than ribosomal proteins, are overwhelmingly targeted. The antibiotics are found at messenger RNA or transfer RNA binding sites and, most importantly, at pivot locations that are key for the structural rearrangements during the molecular mechanical steps in initiation, elongation, or termination of protein synthesis. We focus here on the 30S particle. Structurally, the antibiotics interact in many ways with RNA: (i) only with the phosphate groups (streptomycin); (ii) mainly with bases (hygromycin, spectinomycin); (iii) with a mixture of both (paromomycin, Geneticin); (iv) via magnesium ions (tetracycline) or a protein side chain (streptomycin). The antibiotics can mimic base stacking (pactamycin) or form pseudo-base pairing interactions with ribosomal bases (paromomycin and related aminoglycosides). Resistance strategies (mutations or methylations in rRNA or enzymatic modifications of the antibiotics) can generally be understood on the basis of the intermolecular contacts made between the antibiotics and rRNA residues in the crystal structures. In humans, toxicity of ribosomal antibiotics is most likely due, at least in part, to the sensitivity of mitochondrial ribosomes, since mitochondria evolved from a bacterial ancestor. Antibiotic families (e.g., aminoglycosides) form a set of invariant H-bonds to defined rRNA residues. When such residues are conserved in bacteria, but not in eukaryotes, resistance of eukaryotic ribosomes is observed. The structural knowledge, together with comparative genomic analysis, should allow for the development of new broad-spectrum antibiotics with higher selectivity toward bacterial ribosomes and less toxicity on eukaryotic cytoplasmic and mitochondrial ribosomes.

Aminoglycosides↗
PubMed2006

Binding of neomycin-class aminoglycoside antibiotics to mutant ribosomes with alterations in the A site of 16S rRNA.

Aminoglycoside antibiotics that bind to the aminoacyl-tRNA site (A site) of the ribosome are composed of a common neamine core in which a glycopyranosyl ring is attached to position 4 of a 2-deoxystreptamine moiety. The core is further substituted by one (ribostamycin), two (neomycin and paromomycin), or three (lividomycin A) additional sugars attached to position 5 of the 2-deoxystreptamine. To study the role of rings III, IV, and V in aminoglycoside binding, we used isogenic Mycobacterium smegmatis DeltarrnB mutants carrying homogeneous populations of mutant ribosomes with alterations in the 16S rRNA A site. MICs were determined to investigate drug-ribosome interactions, and the results were compared with that of the previously published crystal structure of paromomycin bound to the ribosomal A site. Our analysis demonstrates that the stacking interaction between ring I and G1491 is largely sequence independent, that rings III and IV each increase the strength of drug binding to the ribosome, that ring IV of the 6'-NH3+ aminoglycosides compensates for loss of interactions between ring II and U1495 and between ring III and G1491, that the aminoglycosides rely on pseudo-base pairing between ring I and A1408 for binding independently of the number of sugar rings attached to the neamine core, that addition of ring V to the 6'-OH 4,5-aminoglycoside paromomycin does not alter the mode of binding, and that alteration of the U1406.U1495 wobble base pair to the Watson-Crick interaction pair 1406C-1495G yields ribosomal drug susceptibilities to 4,5-aminoglycosides comparable to those seen with the wild-type A site.

Anti-Bacterial Agents↗
PubMed2006

Topology of three-way junctions in folded RNAs.

The three-way junctions contained in X-ray structures of folded RNAs have been compiled and analyzed. Three-way junctions with two helices approximately coaxially stacked can be divided into three main families depending on the relative lengths of the segments linking the three Watson-Crick helices. Each family has topological characteristics with some conservation in the non-Watson-Crick pairs within the linking segments as well as in the types of contacts between the segments and the helices. The most populated family presents tertiary interactions between two helices as well as extensive shallow/minor groove contacts between a linking segment and the third helix. On the basis of the lengths of the linking segments, some guidelines could be deduced for choosing a topology for a three-way junction on the basis of a secondary structure. Examples and prediction bas'ed on those rules are discussed.

Base Pairing↗
PubMed2005

Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding.

The crystal structures of six complexes between aminoglycoside antibiotics (neamine, gentamicin C1A, kanamycin A, ribostamycin, lividomycin A and neomycin B) and oligonucleotides containing the decoding A site of bacterial ribosomes are reported at resolutions between 2.2 and 3.0 A. Although the number of contacts between the RNA and the aminoglycosides varies between 20 and 31, up to eight direct hydrogen bonds between rings I and II of the neamine moiety are conserved in the observed complexes. The puckered sugar ring I is inserted into the A site helix by stacking against G1491 and forms a pseudo base pair with two H-bonds to the Watson-Crick sites of the universally conserved A1408. This central interaction helps to maintain A1492 and A1493 in a bulged-out conformation. All these structures of the minimal A site RNA complexed to various aminoglycosides display crystal packings with intermolecular contacts between the bulging A1492 and A1493 and the shallow/minor groove of Watson-Crick pairs in a neighbouring helix. In one crystal, one empty A site is observed. In two crystals, two aminoglycosides are bound to the same A site with one bound specifically and the other bound in various ways in the deep/major groove at the edge of the A sites.

Adenine↗
PubMed2005

Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA folding.

Though the molecular architecture of many native RNA structures has been characterized, the structures of folding intermediates are poorly defined. Here, we present a nucleotide-level model of a highly structured equilibrium folding intermediate of the specificity domain of the Bacillus subtilis RNase P RNA, obtained using chemical and nuclease mapping, circular dichroism spectroscopy, small-angle X-ray scattering and molecular modeling. The crystal structure indicates that the 154 nucleotide specificity domain is composed of several secondary and tertiary structural modules. The structure of the intermediate contains modules composed of secondary structures and short-range tertiary interactions, implying a sequential order of tertiary structure formation during folding. The intermediate lacks the native core and several long-range interactions among peripheral regions, such as a GAAA tetraloop and its receptor. Folding to the native structure requires the local rearrangement of a T-loop in the core in concert with the formation of the GAAA tetraloop-receptor interaction. The interplay of core and peripheral structure formation rationalizes the high degree of cooperativity observed in the folding transition leading to the native structure.

Bacillus subtilis↗
PubMed2005

An mRNA is capped by a 2', 5' lariat catalyzed by a group I-like ribozyme.

Twin-ribozyme introns are formed by two ribozymes belonging to the group I family and occur in some ribosomal RNA transcripts. The group I-like ribozyme, GIR1, liberates the 5' end of a homing endonuclease messenger RNA in the slime mold Didymium iridis. We demonstrate that this cleavage occurs by a transesterification reaction with the joining of the first and the third nucleotide of the messenger by a 2',5'-phosphodiester linkage. Thus, a group I-like ribozyme catalyzes an RNA branching reaction similar to the first step of splicing in group II introns and spliceosomal introns. The resulting short lariat, by forming a protective 5' cap, might have been useful in a primitive RNA world.

Base Sequence↗
PubMed2005

MAO: a Multiple Alignment Ontology for nucleic acid and protein sequences.

The application of high-throughput techniques such as genomics, proteomics or transcriptomics means that vast amounts of heterogeneous data are now available in the public databases. Bioinformatics is responding to the challenge with new integrated management systems for data collection, validation and analysis. Multiple alignments of genomic and protein sequences provide an ideal environment for the integration of this mass of information. In the context of the sequence family, structural and functional data can be evaluated and propagated from known to unknown sequences. However, effective integration is being hindered by syntactic and semantic differences between the different data resources and the alignment techniques employed. One solution to this problem is the development of an ontology that systematically defines the terms used in a specific domain. Ontologies are used to share data from different resources, to automatically analyse information and to represent domain knowledge for non-experts. Here, we present MAO, a new ontology for multiple alignments of nucleic and protein sequences. MAO is designed to improve interoperation and data sharing between different alignment protocols for the construction of a high quality, reliable multiple alignment in order to facilitate knowledge extraction and the presentation of the most pertinent information to the biologist.

Databases, Genetic↗