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E Westhof

Publications and source records attributed to E Westhof.

At least 19 recordsLinked to original sources
PubMed1996

A tyrosyl-tRNA synthetase recognizes a conserved tRNA-like structural motif in the group I intron catalytic core.

The Neurospora crassa mitochondrial (mt) tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns, in addition to aminoacylating tRNA(Tyr). Here, we compared the CYT-18 binding sites in the N. crassa mt LSU and ND1 introns with that in N. crassa mt tRNA(Tyr) by constructing three-dimensional models based on chemical modification and RNA footprinting data. Remarkably, superimposition of the CYT-18 binding sites in the model structures revealed an extended three-dimensional overlap between the tRNA and the group I intron catalytic core. Our results provide insight into how an RNA-splicing factor can evolve from a cellular RNA-binding protein. Further, the structural similarities between group I introns and tRNAs are consistent with an evolutionary relationship and suggest a general mechanism for the evolution of complex catalytic RNAs.

Base Sequence↗
PubMed1996

Structural basis of ligand discrimination by two related RNA aptamers resolved by NMR spectroscopy.

In a previous study, an RNA aptamer for the specific recognition of arginine was evolved from a parent sequence that bound citrulline specifically. The two RNAs differ at only 3 positions out of 44. The solution structures of the two aptamers complexed to their cognate amino acids have now been determined by two-dimensional nuclear magnetic resonance spectroscopy. Both aptamers contain two asymmetrical internal loops that are not well ordered in the free RNA but that fold into a compact structure upon ligand binding. Those nucleotides common to both RNAs include a conserved cluster of purine residues, three of which form an uneven plane containing a G:G pair, and two other residues nearly perpendicular to that surface. Two of the three variant nucleotides are stacked on the cluster of purines and form a triple contact to the amino acid side chain, whereas the edge of the third variant nucleotide is capping the binding pocket.

Arginine↗
PubMed1996

Mapping in three dimensions of regions in a catalytic RNA protected from attack by an Fe(II)-EDTA reagent.

The accessibility of the ribose groups in the phosphodiester chain of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, has been probed with an Fe(II)-EDTA reagent when the RNA is alone in solution, when it is in a complex with a tRNA precursor substrate, and when it is in the holoenzyme complex with its cofactor, C5 protein. The regions found to be protected under these various conditions, as well as those previously identified in other chemical probing experiments, have been mapped on a three-dimensional working model of M1 RNA and are generally compatible with the previously proposed placement of the substrate on the enzyme and with previous data and inferences regarding the interactions of C5 protein with M1 RNA. On the basis of the accessibilities of the C(4') atoms, refinements have been introduced in the model to accommodate the Fe(II)-EDTA protection data. The protein cofactor makes contact with several helical regions of the catalytic RNA on the opposite side of the surface to which substrates bind.

Bacterial Proteins↗
PubMed1996

The environment of two metal ions surrounding the splice site of a group I intron.

Several divalent metal ions (Ca2+, Sr2+ and Pb2+) do not promote splicing, but instead induce cleavage at a single site in the conserved group I intron core in the absence of the guanosine cofactor at elevated pH, generating products with 5'-OH and 3'-phosphate ends. The reaction is competed by Mg2+, which does not cleave at this position, but hydrolyses the splice sites producing 3'-OH and 5'-phosphate ends. Mn2+ promotes both core cleavage and splice site hydrolysis under identical conditions, suggesting that two different metal atoms are involved, each responsible for one type of cleavage, and with different chemical and geometric requirements. Based on the core cleavage position and on the previously proposed coordination sites for Mg2+, we propose a structural location for two metal ions surrounding the splice site in the Michel-Westhof three-dimensional model of the group I intron core. The proposed location was strengthened by a first mutational analysis which supported the suggested interaction between one of the metal ions and the bulged residue in P7.

Bacteriophage T4↗
PubMed1996

Usefulness of functional and structural solution data for the modeling of tRNA-like structures.

Structures of large RNAs are not easily solved by X-ray crystallography or by NMR spectroscopy. This paper reviews the alternate methodology based on enzymatic and chemical mapping data collected on RNAs combined with graphical modeling for the construction of three-dimensional models. The different steps that lead to the establishment of the models are critically discussed. It is shown how the correctness of an RNA model can be strengthened by establishing correlations between the structure and the functionality of the molecule and its variants. Finally, the predictive potential of a model is discussed The approach is illustrated by results obtained on plant viral tRNA-like structures, and particularly on that of brome mosaic virus (BMV) RNA.

Base Sequence↗
PubMed1996

Function of a pseudoknot in the suppression of an alternative splicing event in a group I intron.

Like most mitochondrial group I introns with a free-standing open reading frame (ORF) located downstream of their catalytic core, the Sd.cob, 1 intron in the gene coding for the cytochrome b of Saccharomyces douglasii mitochondria possesses a putative proximal 3' splice site. However, incubation of Sd.cob, 1 preRNA transcripts under optimal in vitro splicing conditions essentially results in splicing at the authentic, distal 3' splice junction. The mechanism by which the proximal splicing event is suppressed in vitro involves formation of a tertiary interaction which is only found in the Sd.cob, 1 intron. Core nucleotides located in loop L5 block proximal splicing by forming Watson-Crick base pairs with the nucleotide sequence of the proximal 3' splice site. This tertiary base pairing, also important for the folding of the intron into an active conformation, may be regarded as equivalent to the L9/P5, GNRA-loop/helix interaction found in more than one-third of known group I introns.

Alternative Splicing↗
PubMed1996

H-bond stability in the tRNA(Asp) anticodon hairpin: 3 ns of multiple molecular dynamics simulations.

Multiple molecular dynamics trajectories of the solvated and neutralized 17-residue tRNA(Asp) anticodon hairpin were generated for a total of 3 ns. Explicit treatment of all long-ranged electrostatic interactions by the particle mesh Ewald algorithm, as implemented in the AMBER MD software package, effected a degree of structural stabilization not previously achieved by use of a long 16-A solvent interaction truncation scheme. The increased stability of this multiple molecular dynamics set was appropriate for an in-depth analysis of the six 500-ps-long trajectories and allowed the characterization of a number of key structural interactions. The dynamical behavior of the standard Watson-Crick base pairs, the noncanonical G30-U40 "wobble" base pair, and the psi 32-C38 pseudo-base pair is presented as well as that of two C--H... O hydrogen bonds found to contribute to the array of tertiary interactions that stabilize the seven-nucleotide native loop conformation. The least mobile residue in the loop is U33, which forms the U-turn motif and which participates in several hydrogen-bonding interactions, whereas the most mobile residue is the apical residue G34 at the wobble position, a factor undoubtedly important in its biological function. The set of multiple molecular dynamics trajectories obtained does not converge on a 500-ps time scale to a unique dynamical model but instead describes an ensemble of structural microstates accessible to the system under the present simulation protocol, which is the result of local structural heterogeneity rather than of global conformational changes.

Anticodon↗
PubMed1996

RNAs mediating cotranslational insertion of selenocysteine in eukaryotic selenoproteins.

Selenocysteine, a selenium-containing analog of cysteine, is found in the prokaryotic and eukaryotic kingdoms in active sites of enzymes involved in oxidation-reduction reactions. Its biosynthesis and cotranslational insertion into selenoproteins is performed by an outstanding mechanism, implying the participation of several gene products. The tRNA(Sec) is one of these. In eukaryotes, its transcription mode by RNA polymerase III differs from that of classical tRNA genes, both at the level of the promoter elements and transcription factors involved. In addition, enhanced transcription is afforded by a newly characterized zinc finger activator. Not only transcription of the gene, but also the tRNA(Sec) itself is atypical since its 2D and 3D structures exhibit features which set it apart from classical tRNAs. Decoding of eukaryotic selenocysteine UGA codons requires a stem-loop structure in the 3'UTR of mRNAs, the selenocysteine insertion sequence (SECIS) element. Structure probing and sequence comparisons led us to propose a 2D structure model for the SECIS element, containing a novel RNA motif composed of four consecutive non-Watson-Crick base-pairs. A 3D model, rationalizing the accessibility data, was elaborated by computer modeling. It yields indicative or suggestive evidence for the role that could play some conserved residues and/or structural features in SECIS function. These might act as signals for interaction with SBP, the SECIS binding protein that we have characterized.

Animals↗
PubMed1996

Calculations of nucleic acid conformations.

The present computational power and sophistication of theoretical approaches to nucleic acid structural investigation are sufficient for the realization of static and dynamic models that correlate accurately with current crystallographic, NMR and solution-probing structural data, and consequently are able to provide valuable insights and predictions for a variety of nucleic acid conformational families. In molecular dynamics simulations, the year 1995 was marked by the foray of fast Ewald methods, an accomplishment resulting from several years' work in the search for an adequate treatment of the electrostatic long-range forces so primordial in nucleic acid behavior. In very large systems, and particularly in the RNA-folding field, techniques originating from artificial intelligence research, like constraint satisfaction programming or genetic algorithms, have established their utility and potential.

DNA↗
PubMed1996

New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme.

BACKGROUND: Group I introns self-splice via two consecutive trans-esterification reactions in the presence of guanosine cofactor and magnesium ions. Comparative sequence analysis has established that a catalytic core of about 120 nucleotides is conserved in all known group I introns. This core is generally not sufficient for activity, however, and most self-splicing group I introns require non-conserved peripheral elements to stabilize the complete three-dimensional (3D) structure. The physico-chemical properties of group I introns make them excellent systems for unraveling the structural basis of the RNA-RNA interactions responsible for promoting the self-assembly of complex RNAs. RESULTS: We present phylogenetic and experimental evidence for the existence of three additional tertiary base pairings between hairpin loops within peripheral components of subgroup IC1 and ID introns. Each of these new long range interactions, called P13, P14 and P16, involves a terminal loop located in domain 2. Although domains 2 of IC and ID introns share very strong sequence similarity, their terminal loops interact with domains 5 and 9 (subgroup IC1) and domain 6 (subgroup ID). Based on these tertiary contacts, comparative sequence analysis, and published experimental results such as Fe(II)-EDTA protection patterns, we propose 3D models for two entire group I introns, the subgroup IC1 intron in the large ribosomal precursor RNA of Tetrahymena thermophila and the SdCob.1 subgroup ID intron found in the cytochrome b gene of Saccharomyces douglasii. CONCLUSIONS: Three-dimensional models of group I introns belonging to four different subgroups are now available. They all emphasize the modular and hierarchical organization of the architecture of group I introns and the widespread use of base-pairings between terminal hairpin loops for stabilizing the folded and active structures of large and complex RNA molecules.

Animals↗
PubMed1996

A central pseudoknotted three-way junction imposes tRNA-like mimicry and the orientation of three 5' upstream pseudoknots in the 3' terminus of tobacco mosaic virus RNA.

A three-dimensional model of the histidylable 3'-terminal tRNA-like domain of tobacco mosaic virus RNA is proposed on the basis of a comparative structural analysis, chemical and enzymatic probing, combined with graphical modeling of three RNA constructs of increasing size (38, 108, and 182 nt) derived from the 3'-terminal viral RNA sequence. The comparison between the probing patterns of the three RNAs allowed the determination of the relative orientation of these structural domains in the full-length viral tRNA-like structure. Modeling data indicate that only one of the two possible isomers of the three-way junction located at a central position of the tRNA-like domain is in agreement with structural data. Interestingly, this isomer gives rise to a molecule bearing a structural mimicry with the L-shape of canonical tRNAs. A pseudoknotted acceptor branch containing a T-like loop is located perpendicularly to an anticodon-like branch. Moreover, a single-stranded RNA stretch belonging to the pseudoknotted central core mimics a D-like loop and it is proposed that it interacts via two conserved guanosines with nucleotides of the T-like loop as found in canonical tRNAs. This model is valid for the 3' noncoding regions of tobamoviral RNAs as well as for the tRNA-like domain of the satellite tobacco mosaic virus RNA. All three molecules are substrates for yeast HisRS; however, whereas the complete viral genome is required for optimal histidylation capacities, both charging levels and affinity constants are decreased for the three RNA transcripts, suggesting that additional contacts located outside the tRNA-like domain are needed for an optimal aminoacylation process.

Base Sequence↗
PubMed1996

Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SELB.

On the basis of chemical probing data, the solution structures of RNA hairpins within fdhF and fdnG mRNAs in Escherichia coli, which both promote selenocysteine incorporation at UGA codons, were derived with the help of computer modeling. We find that these mRNA hairpins contain two separate structural domains that possibly also exert two different functions. The first domain is comprised of the UGA codon, which is included within a complex and distorted double-stranded region. Thereby, release factor 2 might be prevented from binding to the UGA codon to terminate protein synthesis. The second domain is located within the apical loop of the mRNA hairpin structures. This loop region exhibits a defined tertiary structure in which no base is involved in Watson-Crick interactions. The structure of the loop is such that, following a sharp turn after G22 (A22 in fdnG mRNA), bases G23 and U24 are exposed to the solvent on the deep groove side of the supporting helix. Residues C25 and U26 close the loop with a possible single H-bonding interaction between the first and last residues of the loop, 04(U26) and N6(A21). The bulge residues U17 and U18 (in fdhF mRNA), or Ul7 only in fdnG mRNA, point their Watson-Crick positions in the same direction as loop residues G23 and U24 do, and at the same time open up the deep groove at the top of the hairpin helix. Chemical probing data demonstrate that bases G23 and U24 in both mRNA hairpins, as well as residues U17 and Ul7/U18 (for fdhF mRNA) located in a bulge 5' to the loop, are involved directly in binding to special elongation factor SELB in both mRNAs. Therefore, SELB recognizes identical bases within both mRNA hairpins despite differences in their primary sequence, consistent with the derived 3D models for these mRNAs, which exhibit similar tertiary structures. Binding of SELB to the fdhF mRNA hairpin was estimated to proceed with an apparent Kd of 30 nM.

Aldehydes↗
PubMed1996

A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs.

In eukaryotes, co-translational insertion of selenocysteine into selenoproteins necessitates the participation of the selenocysteine insertion sequence (SECIS), an element lying in the 3'-untranslated region of selenoprotein mRNAs. We report a detailed experimental study of the secondary structures of the SECIS elements of three selenoprotein mRNAs, the rat and human type I iodothyronine deiodinase (5'DI) and rat glutathione peroxidase (GPx). Based on RNase and chemical probing, a new secondary structure model is established. It is characterized by a stem-loop structure, comprising two helices (I and II) separated by an internal loop, with an apical loop surmounting helix II. Sequence comparisons of 20 SECIS elements, arising from 2 5'DI, 13 GPx, 2 selenoprotein P, and 1 selenoprotein W mRNAs, confirm the secondary structure model. The most striking finding of the experimental study concerns a set of conserved sequences in helix II that interact to form a novel RNA structural motif consisting of a quartet composed of non-Watson-Crick base pairs 5'UGAY3': 5'UGAU3'. The potential for forming the quartet is preserved in 15 SECIS elements, but three consecutive non-Watson-Crick base pairs can nevertheless form in the other five SECIS, the central G.A tandem being invariant in all cases. A 3D model, derived by computer modeling with the use of the solution data, suggests that the base pairing interactions in the G.A tandem are of the type found in GNRA loops. The 3D model displays the quartet lying in an accessible position at the foot of helix II, which is bent at the internal loop, suggesting that the non-Watson-Crick base pair arrangement provides an unusual pattern of chemical groups for putative ligand interaction.

Animals↗
PubMed1996

The 16S rRNA binding site of Thermus thermophilus ribosomal protein S15: comparison with Escherichia coli S15, minimum site and structure.

Binding of Escherichia coli and Thermus thermophilus ribosomal proteins S15 to a 16S ribosomal RNA fragment from T. thermophilus (nt 559-753) has been investigated in detail by extensive deletion analysis, filter-binding assays, gel mobility shift, structure probing, footprinting with chemical, enzymatic, and hydroxyl radical probes. Both S15 proteins recognize two distinct sites. The first one maps in the bottom of helix 638-655/717-734 (H22) and in the three-way junction between helix 560-570/737-747 (H20), helix 571-600/606-634 (H21), and H22. The second is located in a conserved purine-rich region in the center of H22. The first site provides a higher contribution to the free energy of binding than the second one, and both are required for efficient binding. A short RNA fragment of 56 nt containing these elements binds S15 with high affinity. The structure of the rRNA is constrained by the three-way junction and requires both magnesium and S15 to be stabilized. A 3D model, derived by computer modeling with the use of experimental data, suggests that the bound form adopts a Y-shaped conformation, with a quasi-coaxial stacking of H22 on H20, and H21 forming an acute angle with H22. In this model, S15 binds to the shallow groove of the RNA on the exterior side of the Y-shaped structure, making contact with the two sites, which are separated by one helix turn.

Base Sequence↗
PubMed1995

An interactive framework for RNA secondary structure prediction with a dynamical treatment of constraints.

A novel approach aiding in the prediction of RNA secondary structures is presented. Although phylogenetic methods are the most successful at deriving RNA secondary structures, the are not applicable when the number of sequences or the sequence variability is too low. Methods based on energy minimization are therefore of great interest. However, some of the suboptimal RNA secondary structures computed with classic methods are unsaturated structures, i.e. some structures are included into others. Thus, the incorporation of constraints during the process of folding is not possible, while the incorporation of constraints before the process of folding often introduces a bias into the energy function. This paper describes a new procedure which allows for the incorporation of constraints before and during the process of RNA folding. SAPSSARN is an interactive program which offers a framework, both to specify a secondary structure through a set of folding constraints and to compute all the supoptimal saturated RNA secondary structures which satisfy all the folding constraints. At the start, it relies on the computation of the probabilities of pairing of each base with all others according to McCaskill's algorithm. The constraint satisfaction formulation of the problem deals dynamically with a chosen set of folding constraints and, finally, a search algorithm computes all the suboptimal saturated secondary structures which satisfy those folding constraints. Within such a framework, it is possible to test new ideas about RNA folding and secondary structures, including pseudoknots, can be computed. The program is illustrated with RNA sequences on which we obtained results in agreement with known structures by using a protocol which mimics the hierarchical folding of RNA molecules.

Algorithms↗
PubMed1995

Molecular dissection of the pseudoknot governing the translational regulation of Escherichia coli ribosomal protein S15.

The ribosomal protein S15 controls its own translation by binding to a mRNA region overlapping the ribosome binding site. That region of the mRNA can fold in two mutually exclusive conformations that are in dynamic equilibrium: a structure with two hairpins and a pseudoknot. A mutational analysis provided evidence for the existence and requirement of the pseudoknot for translational control in vivo and S15 recognition in vitro. In this study, we used chemical probing to analyze the structural consequences of mutations and their effect on the stem-loop/pseudoknot equilibrium. Interactions between S15 and the pseudoknot structure were further investigated by footprinting experiments. These data, combined with computer modelling and the previously published data on S15 binding and in vivo control, provide important clues on pseudoknot formation and S15 recognition. An unexpected result is that the relevant control element, here the pseudoknot form, can exist in a variety of topologically equivalent structures recognizable and shapable by S15. S15 sits on the deep groove of the co-axial stack and makes contacts with both stems, shielding the bridging adenine. The only specific sequence determinants are found in the helix common to the pseudoknot and the hairpin structures.

Base Sequence↗