SEARCH · PubMed Health

Results for “RNA, Transfer, Asp”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1999

Molecular dynamics simulations of solvated yeast tRNA(Asp).

Transfer RNA molecules are involved in a variety of biological processes, implying complex recognition events with proteins and other RNAs. From a structural point of view, tRNAs constitute a reference system for studying RNA folding and architecture. A deeper understanding of their structural and functional properties will derive from our ability to model accurately their dynamical behavior. We present the first dynamical model of a fully neutralized and solvated tRNA molecule over a 500-ps time scale. Starting from the crystallographic structure of yeast tRNA(Asp), the 75-nucleotide molecule was modeled with 8055 water molecules and 74 NH4+ counterions, using the AMBER4.1 program and the particle mesh Ewald (PME) method for the treatment of long-range electrostatic interactions. The calculations led to a dynamically stable model of the tRNA molecule. During the simulation, all secondary and tertiary base pairs are maintained while a certain lability of base triples in the tRNA core is observed. This lability was interpreted as resulting from intrinsic factors associated with the "weaker" hydrogen bonding patterns seen in these base triples and from an altered ionic environment of the tRNA molecule. Calculated thermal factors are used to compare the dynamics of the tRNA in solution and in the crystal. The present molecular dynamics simulation of a complex and highly charged nucleic acid molecule attests to the fact that simulation methods are now able to investigate not only the dynamics of proteins, but also that of large RNA molecules. Thus they also provide a basis for further investigations on the structural and functional effects of chemical and posttranscriptionally modified nucleotides as well as on ionic environmental effects.

Base Pairing↗

PubMed1988

Restrained refinement of two crystalline forms of yeast aspartic acid and phenylalanine transfer RNA crystals.

Four transfer RNA crystals, the monoclinic and orthorhombic forms of yeast tRNA(Phe) as well as forms A and B of yeast tRNA(Asp), have been submitted to the same restrained least-squares refinement program and refined to an R factor well below 20% for about 4500 reflections between 10 and 3 A. In yeast tRNA(Asp) crystals the molecules exist as dimers with base pairings of the anticodon (AC) triplets and labilization of the tertiary interaction between one invariant guanine of the dihydrouridine (D) loop and the invariant cytosine of the thymine (T) loop (G19-C56). In yeast tRNA(Phe) crystals, the molecules exist as monomers with only weak intermolecular packing contacts between symmetry-related molecules. Despite this, the tertiary folds of the L-shaped tRNA structures are identical when allowance is made for base sequence changes between tRNA(Phe) and tRNA(Asp). However, the relative mobilities of two regions are inverse in the two structures with the AC loop more mobile than the D loop in tRNA(Phe) and the D loop more mobile than the AC loop in tRNA(Asp). In addition, the T loop becomes mobile in tRNA(Asp). The present refinements were performed to exclude packing effects or refinement bias as possible sources of such differential dynamic behavior. It is concluded that the transfer of flexibility from the anticodon to the D- and T-loop region in tRNA(Asp) is not a crystal-line artefact. Further, analysis of the four structures supports a mechanism for the flexibility transfer through base stacking in the AC loop and concomitant variations in twist angles between base pairs of the anticodon helix which propagate up to the D- and T-loop region.

Anticodon↗

PubMed2004

A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon.

Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA(Asp) QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA(Asp) anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved among prokaryotes.

Acylation↗

PubMed1992

Effect of conformational features on the aminoacylation of tRNAs and consequences on the permutation of tRNA specificities.

The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.

Aspartate-tRNA Ligase↗

PubMed2006

A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine.

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNAAsn and tRNAGln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNAAsn and Gln-tRNAGln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNAAsn and tRNAAsp variants, that amidation of Asp acylating tRNAAsn is promoted by the base pair U1-A72 whereas the G1-C72 pair and presence of the supernumerary nucleotide U20A in the D-loop of tRNAAsp prevent amidation. We predict, based on comparison of tRNAGln and tRNAGlu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNAGln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G46 and U47 of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.

Adenine↗

PubMed1996

RNA editing changes the identity of a mitochondrial tRNA in marsupials.

In the mitochondrial genome of marsupials, the tRNA gene located at the position where in other mammals an aspartyl-tRNA is encoded carries the glycine anticodon GCC. Post-transcriptionally, an RNA editing mechanism affects the second position of the anticodon such that the aspartate anticodon GUC is created in approximately 50% of the mature tRNA pool. We show that the unedited version of this tRNA'Asp' (GCC) can be specifically aminoacylated with glycine in vitro, while the edited version becomes aminoacylated with aspartic acid. Furthermore, we show that both forms are aminoacylated to a substantial extent in vivo. By replacing an amino group with a keto group, RNA editing thus changes the identity of this tRNA allowing a single gene to encode two tRNAs.

Animals↗

PubMed1997

Structure and aminoacylation capacities of tRNA transcripts containing deoxyribonucleotides.

The contribution of the ribose 2'-hydroxyls to RNA structure and function has been analyzed, but still remains controversial. In this work, we report the use of a mutant T7 RNA polymerase as a tool in RNA studies, applied to the aspartate and methionine tRNA aminoacylation systems from yeast. Our approach consists of determining the effect of substituting natural ribonucleotides by deoxyribonucleotides in RNA and, thereby, defining the subset of important 2'-hydroxyl groups. We show that deoxyribose-containing RNA can be folded in a global conformation similar to that of natural RNA. Melting curves of tRNAs, obtained by temperature-gradient gel electrophoresis, indicate that in deoxyribo-containing molecules, the thermal stability of the tertiary network drops down, whereas the stability of the secondary structure remains unaltered. Nuclease footprinting reveals a significant increase in the accessibility of both single- and double-stranded regions. As to the functionality of the deoxyribose-containing tRNAs, their in vitro aminoacylation efficiency indicates striking differential effects depending upon the nature of the substituted ribonucleotides. Strongest decrease in charging occurs for yeast initiator tRNA(Met) transcripts containing dG or dC residues and for yeast tRNA(Asp) transcripts with dU or dG. In the aspartate system, the decreased aminoacylation capacities can be correlated with the substitution of the ribose moieties of U11 and G27, disrupting two hydrogen bond contacts with the synthetase. Altogether, this suggests that specific 2'-hydroxyl groups in tRNAs can act as determinants specifying aminoacylation identity.

Base Sequence↗

PubMed1997

The complete mitochondrial genome of the wallaroo (Macropus robustus) and the phylogenetic relationship among Monotremata, Marsupialia, and Eutheria.

The complete mitochondrial DNA (mtDNA) (16,896 nt) of the wallaroo (Macropus robustus) was sequenced. The concatenated amino acid sequences of 12 mitochondrial protein-coding genes of the wallaroo plus those of a number of other mammals were included in a phylogenetic study of early mammalian divergences. The analysis joined monotremes and marsupials (the Marsupionta hypothesis) to the exclusion of eutherians. The analysis rejected significantly the commonly acknowledged Theria hypothesis, according to which Marsupialia and Eutheria are grouped together to the exclusion of Monotremata. The region harboring the gene for lysine tRNA (tRNA-Lys) in the mtDNA of other vertebrates is in the wallaroo occupied by a sequence (tRNA-Lys) that lacks both an anticodon loop as well as the anticodon for the amino acid lysine. An alternative tRNA-Lys gene was not identified in any other region of the mtDNA of the wallaroo, suggesting that a tRNA-Lys of nuclear origin is imported into marsupial mitochondria. Previously described RNA editing of tRNA-Asp and rearrangement of some tRNA genes were reconfirmed in the mtDNA of the wallaroo. The divergence between Monotremata/Marsupialia and Eutheria was timed to approximately 130 million years before present (MYBP). The same calculations suggested that Monotremata and Marsupialia diverged approximately 115 MYBP and that Australian and American marsupials separated approximately 75 MYBP. The findings also show that many, probably most, extant eutherian orders had their origin in middle to late Cretaceous times, 115-65 MYBP.

Animals↗

PubMedDate not supplied

[Chemical ribonucleases. 4. Analysis of the fragment structure of chemical ribonucleases based on 1,4-diazabicyclo[2.2.2]octane].

Artificial ribonucleases of the ABLkCm series were synthesized. They consist of a lipophilic alkyl radical (Et, n-C14H29, or C15H31) A, an "RNA-binding domain" B (bisquaternary salt of 1,4-diazabicyclo[2.2.2]octane), a "catalytic domain" Cm [histamine (C1) or histidine (C3) residue], and a "linker" Lk that joins the "domains" B and Cm [here, k is the number of methylene units (one or three) in the linker]. The effect of the "domain structure" on the catalytic properties of the chemical ribonucleases was analyzed using seven compounds of this series (ABL1C1, ABL3C1, ABL3C3, AC1, AB, BL2, and BL3C3). The catalytic activity of the compounds was assessed in the reaction of hydrolysis of the in vitro transcripts of human tRNA(Lys) and yeast tRNA(Asp) under physiological conditions. It was shown that only chemical ribonucleases that involve all the fragments of the ABLkCm construct can hydrolyze the substrate tRNA at a high rate (90% of tRNA is hydrolyzed for 10 h at 37 degrees C). The activity of the compounds is largely determined by the presence of a long lipophilic radical linked to 1,4-diazabicyclo[2.2.2]octane and a long linker, which joins the RNA-hydrolyzing and RNA-binding fragments. The results indicate an important role of hydrophobic interactions in the acceleration of the RNA hydrolysis reaction. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

Catalysis↗

PubMed1995

Proton exchange and basepair kinetics of yeast tRNA(Phe) and tRNA(Asp1).

Nuclear magnetic resonance measurements of proton exchange were performed on yeast tRNA(Phe) and yeast tRNA(Asp), at temperatures from 20 to 45 degrees C, in the presence of various levels of salt, phosphate, added magnesium, and pH. The dynamical changes of the tRNA molecule were interpreted, with the aid of firmly established assignments and the use of the saturation recovery technique. In tRNA(Phe), the exchange rates in zero magnesium indicated early melting of the acceptor stem, tertiary structure, and D stem. However, in the presence of even low levels of magnesium the D stem remained intact up to high temperature, stabilized by a Mg2+ ion. A similar unfolding sequence was observed in tRNA(Asp). The difference between these two tRNAs was the thermal behavior of the tertiary resonance U8-A14. In tRNA(Phe), this base pair showed sharp rate increases between 32 and 39 degrees C. However, in tRNA(Asp), it remained intact up to 36 degrees C and disappeared at 39 degrees C, even if there was not important kinetic broadening. By measuring the temperature dependence of the exchange rates, we obtained an activation energy of 40-60 kcal/mol for all the imino protons of yeast tRNA(Phe) in zero magnesium. The same activation energy was obtained for tRNA(Phe) with equimolar concentration of magnesium. By investigation of the dependence of the exchange rates of these imino protons on solution conditions, we observed the transition from kop rate limiting in the absence of magnesium to kex rate limiting in the presence of magnesium.

Base Composition↗

PubMed1995

Structural specificity of nuclease from wheat chloroplasts stroma.

A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.

Base Sequence↗

PubMed2001

The polypeptide chain release factor eRF1 specifically contacts the s(4)UGA stop codon located in the A site of eukaryotic ribosomes.

It has been shown previously [Brown, C.M. & Tate, W.P. (1994) J. Biol. Chem. 269, 33164-33170.] that the polypeptide chain release factor RF2 involved in translation termination in prokaryotes was able to photocrossreact with mini-messenger RNAs containing stop signals in which U was replaced by 4-thiouridine (s4U). Here, using the same strategy we have monitored photocrosslinking to eukaryotic ribosomal components of 14-mer mRNA in the presence of tRNA(f)(Met), and 42-mer mRNA in the presence of tRNA(Asp) (tRNA(Asp) gene transcript). We show that: (a) both 14-mer and 42-mer mRNAs crossreact with ribosomal RNA and ribosomal proteins. The patterns of the crosslinked ribosomal proteins are similar with both mRNAs and sensitive to ionic conditions; (b) the crosslinking patterns obtained with 42-mer mRNAs show characteristic modification upon addition of tRNA(Asp) providing evidence for appropriate mRNA phasing onto the ribosome. Similar changes are not detected with the 14-mer mRNA.tRNA(f)(Met) pairs; (c) when eukaryotic polypeptide chain release factor 1 (eRF1) is added to the ribosome.tRNA(Asp) complex it crossreacts with the 42-mer mRNA containing the s(4)UGA stop codon located in the A site, but not with the s(4)UCA sense codon; this crosslink involves the N-terminal and middle domains of eRF1 but not the C domain which interacts with eukaryotic polypeptide chain release factor 3 (eRF3); (d) addition of eRF3 has no effect on the yield of eRF1-42-mer mRNA crosslinking and eRF3 does not crossreact with 42-mer mRNA. These experiments delineate the in vitro conditions allowing optimal phasing of mRNA on the eukaryotic ribosome and demonstrate a direct and specific contact of 'core' eRF1 and s(4)UGA stop codon within the ribosomal A site.

Animals↗

PubMed2000

Phylogenetic relationships of the North American sturgeons (order Acipenseriformes) based on mitochondrial DNA sequences.

The evolutionary relationships of the extant species within the order Acipenseriformes are not well understood. Nucleotide sequences of four mitochondrial genes (12S rRNA, COII, tRNA(Phe), and tRNA(Asp) genes) in North American sturgeon and paddlefish were examined to reconstruct a phylogeny. Analysis of the combined gene sequences suggests a basal placement of the paddlefish with regard to the sturgeons. Nucleotide sequences of all four genes for the three Scaphirhynchus species were identical. The position of Scaphirhynchus based on our data was uncertain. Within the genus Acipenser, the two Acipenser oxyrinchus subspecies were very similar in sequence and found to be basal to the remaining Acipenser species examined. Based on our data, Acipenser transmontanus and Acipenser medirostris were sister taxa, as were Acipenser fulvescens and Acipenser brevirostrum. Comparison of our results with hypotheses of sturgeon relationships proposed by previous authors is presented. The sequence data presented here are phylogenetically useful and provide a solid foundation of genetic information for the North American Acipenseriformes that can be expanded to include Eurasian species to provide a global picture of sturgeon evolution.

Animals↗

PubMed2000

Evaluation of uranyl photocleavage as a probe to monitor ion binding and flexibility in RNAs.

In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in five RNA molecules with known X-ray structures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA(Phe) and tRNA(Asp) from yeast. Uranyl photocleavage was observed at specific positions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl(2). Uranyl photocleavage correlates well with sites of low calculated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in flexible and open to the solvent regions that can undergo induced-fit without disrupting the native fold. Uranyl photocleavage was compared to N-ethyl-N-nitrosourea and lead-induced cleavages in the context of the three-dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at flexible and accessible sites and correlate with the unspecific cleavages prevalent in dynamic and open regions. Applied in a magnesium-dependent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-affinity metal ion environments, as well as regions involved in conformational transitions.

Animals↗

PubMed1992

Recognition of tertiary structure in tRNAs by Rh(phen)2phi3+, a new reagent for RNA structure-function mapping.

With photoactivation Rh(phen)2phi3+ promotes strand cleavage at sites of tertiary interaction in tRNA. The rhodium complex, which binds double-helical DNA by intercalation in the major groove, yields no cleavage in double-helical regions of the RNA or in unstructured single-stranded regions. Instead, Rh(phen)2phi3+ appears to target regions which are structured so that the major groove is open and accessible for stacking with the complex, as occurs where bases are triply bonded. So as to examine the specificity of this novel reagent and to evaluate its use in probing structural changes in RNAs, cleavage studies have been conducted on two structurally characterized tRNAs, tRNA(Phe) and tRNA(Asp) from yeast, the unmodified yeast tRNA(Phe) transcript, and a chemically modified tRNA(Phe), as well as on a series of tRNA(Phe) mutants. On tRNA(Phe) strong cleavage is observed at residues G22, G45, U47, psi 55, and U59; weaker cleavage is observed at A44, m7G46, and C48. On tRNA(Asp) cleavage is found at residues A21 through G26, psi 32, and U48, with minor cleavage apparent at A44, G45, A46, psi 55, U59, and U60. There is a striking similarity in cleavage observed on these tRNAs, and the sites of cleavage mark regions of tertiary folding. Cleavage on the unmodified tRNA(Phe) transcript resembles closely that found on native yeast tRNA(Phe), but additional sites, primarily in the anticodon loop and stem, are evident. The results indicate that globally the structures containing or lacking the modified bases appear to be the same; the differences in cleavage observed may reflect a loosening or alteration in the structure due to the absence of the modified bases. Cleavage results on mutants of tRNA(Phe) illustrate Rh(phen)2phi3+ as a sensitive probe in characterizing tRNA tertiary structure. Results are consistent with other assays for structural or functional changes. Uniquely, Rh(phen)2phi3+ appears to target directly sites of tertiary interaction. Cleavage results on mutants which involve base changes within the triply bounded region of the molecule indicate that it is the structure of the triply bonded array rather than the individual nucleotides which are being targeted. Chemical modification to promote selective depurination of the third base (m7G46) involved in the triple in the folded, native tRNA leads to the reduction of cleavage by the metal complex; this result shows directly the importance of the stacked triple base structure for recognition by the metal complex.(ABSTRACT TRUNCATED AT 400 WORDS)

2,2'-Dipyridyl↗

PubMed1996

Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease.

OBJECTIVE: To identify and characterize a novel autoantibody, anti-WS, that binds total transfer RNA (tRNA). METHODS: Serum from patient WS, who had polyarthritis, Sjögren's syndrome, Raynaud's phenomenon, and interstitial pulmonary fibrosis, was used in this study. Characteristics of anti-WS and antibody-reactive determinants of tRNA were investigated by 32P immunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro. RESULTS: WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence. 32P immunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8S and 5S ribosomal RNAs from 32P-labeled HeLa cell extract. When deproteinized RNA was used as antigen source, total tRNAs were still precipitated by WS serum. An immunoprecipitation study, using various deletion mutants of Escherichia coli tRNA, demonstrated that both D and T psi C loops were needed for antibody binding. Substitution of nucleotide 18G with 18A of E coli tRNA(Trp), which is essential in the formation of the tertiary "L" shape of tRNA, inhibited binding by anti-WS antibodies. CONCLUSION: Anti-WS antibodies are novel autoantibodies directed against tRNAs. The antibody binding site is the common L-shaped tertiary structure conformed by the D loop and T psi C loop of tRNA, suggesting that the antibodies are induced by a conserved sequence among all species. Furthermore, these antibodies could be a marker for a newly recognized subset of connective tissue disease.

Autoantibodies↗

PubMed1995

Identification of base-triples in RNA using comparative sequence analysis.

Comparative sequence analysis has proven to be a very efficient tool for the determination of RNA secondary structure and certain tertiary interactions. However, base-triples, an important RNA structural element, cannot be predicted accurately from sequence data. We show here that the poor base correlations observed at base-triple positions are the result of two factors. (1) Base covariation is not as strictly required in triples as it is in Watson-Crick pairs. (2) Base-triple structures are less conserved among homologous molecules. A particularity of known triple-helical regions is the presence of multiple base correlations that do not reflect direct pairing. We suggest that natural mutations in base-triples create structural changes that require compensatory mutations in adjacent base-pairs and triples to maintain the triple-helix conformation. On the basis of these observations, we devised two new measures of association that significantly enhance the base-triple signal in correlation studies. We evaluated correlations between base-pairs and single stranded bases, and correlations between adjacent base-pairs. Positions that score well in both analyses are the best triple candidates. This procedure correctly identifies triples, or interactions very close to the proposed triples, in type I and type II tRNAs and in the group I intron.

Base Composition↗

PubMed1998

Vibrational dynamics of transfer RNAs: comparison of the free and synthetase-bound forms.

The vibrational dynamics of transfer RNAs, both free, and complexed with the cognate synthetase, are analyzed using a model (Gaussian network model) which recently proved to satisfactorily describe the collective motions of folded proteins. The approach is similar to a normal mode analysis, with the major simplification that no residue specificity is taken into consideration, which permits us (i) to cast the problem into an analytical form applicable to biomolecular systems including about 10(3 )residues, and (ii) to acquire information on the essential dynamics of such large systems within computational times at least two orders of magnitude shorter than conventional simulations. On a local scale, the fluctuations calculated for yeast tRNAPhe and tRNAAsp in the free state, and for tRNAGln complexed with glutaminyl-tRNA synthetase (GlnRS) are in good agreement with the corresponding crystallographic B factors. On a global scale, a hinge-bending region comprising nucleotides U8 to C12 in the D arm, G20 to G22 in the D loop, and m7G46 to C48 in the variable loop (for tRNAPhe), is identified in the free tRNA, conforming with previous observations. The two regions subject to the largest amplitude anticorrelated fluctuations in the free form, i.e. the anticodon region and the acceptor arm are, at the same time, the regions that experience the most severe suppression in their flexibilities upon binding to synthetase, suggesting that their sampling of the conformational space facilitates their recognition by the synthetase. Likewise, examination of the global mode of motion of GlnRS in the complex indicates that residues 40 to 45, 260 to 270, 306 to 314, 320 to 327 and 478 to 485, all of which cluster near the ATP binding site, form a hinge-bending region controlling the cooperative motion, and thereby the catalytic function, of the enzyme. The distal beta-barrel and the tRNA acceptor binding domain, on the other hand, are distinguished by their high mobilities in the global modes of motion, a feature typical of recognition sites, also observed for other proteins. Most of the conserved bases and residues of tRNA and GlnRS are severely constrained in the global motions of the molecules, suggesting their having a role in stabilizing and modulating the global motion.

Amino Acyl-tRNA Synthetases↗