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R Giegé

Publications and source records attributed to R Giegé.

At least 127 records · Page 7Linked to original sources
PubMed1987

3-D graphics modelling of the tRNA-like 3'-end of turnip yellow mosaic virus RNA: structural and functional implications.

The tRNA-like structure of the aminoacylatable 3'-end of turnip yellow mosaic virus (TYMV) RNA was submitted to 3-D graphics modelling. A model of this structure has been inferred previously from both biochemical results and sequence comparisons which presents a new RNA folding feature, the "pseudoknot". It has been verified that this structure can be constructed without compromising accepted RNA stereochemical rules, namely base stacking and preferential 3'-endo sugar pucker. The model has aided interpretation of previous structural mapping experiments using chemical and enzymatic probes, and new accessibilities of residues could be predicted and tested. Pseudoknots have been considered as potential splice sites because they form antiparallel helical segments in a single RNA molecule. We have examined this possibility with the constructed 3-D model and could verify the hypothesis on a structural basis. The model presents a striking similarity with canonical tRNA and allows a valuable comparison between the protection patterns of yeast tRNA(Val) and tRNA-like viral RNA by cognate yeast valyl-tRNA synthetase against structural probes.

Base Sequence↗
PubMed1986

Contact areas of the turnip yellow mosaic virus tRNA-like structure interacting with yeast valyl-tRNA synthetase.

The tRNA-like structure of turnip yellow mosaic virus is known to be efficiently recognized and aminoacylated by valyl-tRNA synthetase. The present work reports domains in the isolated tRNA-like fragment (159 terminal nucleotides at the 3'-end of the two viral RNAs) in contact with purified yeast valyl-tRNA synthetase. These domains were determined in protection experiments using chemical and enzymatic structural probes. In addition, new data, re-enforcing the validity of the tertiary folding model for the native RNA, are given. In particular, at the level of the amino acid accepting arm it was found that the two phosphate groups flanking the three guanine residues of loop I are inaccessible to ethylnitrosourea. This is in agreement with a higher-order structure of this loop involving "pseudo knotting", as proposed by Rietveld et al. (1982). Valyl-tRNA synthetase efficiently protects the viral RNA against digestion by single-strand-specific S1 nuclease at the level of the anticodon loop. With cobra venom ribonuclease, specific for double-stranded regions of RNA, protection was detected on both sides of the anticodon arm and at the 5'-ends of loop I, a region that is involved in the building up of the acceptor arm. Loop II, which is topologically homologous to the T-loop of canonical tRNA was likewise protected. Weak protection was observed between arms I and II, and at the 3'-side of arm V. This arm, located at the 5'-side of arm IV (homologous to the D-arm of tRNA), does not participate in the pseudo-knotted model of the valine acceptor arm. Ethylnitrosourea was used to determine the phosphates of the tRNA-like structure in close contact with the synthetase. These are grouped in several stretches scattered over the RNA molecule. In agreement with the nuclease digestion results, protected phosphates are located in arms I, II, and III. Additionally, this chemical probe permits detection of other protected phosphates on the 3'-side of arm IV and on both sides of arm V. When displayed in the three-dimensional model of the tRNA-like structure, protected areas are localized on both limbs of the L-shaped RNA. It appears that valyl-tRNA synthetase embraces the entire tRNA-like structure. This is reminiscent of the interaction model of canonical yeast tRNAVal with its cognate synthetase.

Alkylation↗
PubMed1986

Anticodon-anticodon interaction induces conformational changes in tRNA: yeast tRNAAsp, a model for tRNA-mRNA recognition.

The crystal structure of yeast tRNAAsp enables visualization of an anticodon-anticodon interaction at the molecular level. Except for differences in the base stacking and twist, the overall conformation of the anticodon loop is quite similar to that of yeast tRNAPhe. The anticodon nucleotide triplets, GUC, of two symmetrically related molecules form a minihelix of the RNA type 11. The modified base m1G37 stacks on both sides of the triplets and enforces the continuity with the anticodon stems. Anticodon association induces long-range conformational changes in the region of the dihydrouracil and thymine loops. Experimental evidence includes the variation in the distribution of temperature factors between yeast tRNAPhe and tRNAAsp, the difference in the self-splitting patterns of tRNAAsp in crystal and solution, and the differential accessibility of cytidines to dimethyl sulfate in free and duplex tRNAAsp. These observations are linked to the fragility and disruption of the G.C Watson-Crick base pair at the corner of the molecule formed by the dihydrouracil and thymine loops.

Anticodon↗
PubMed1986

Studies on anticodon-anticodon interactions: hemi-protonation of cytosines induces self-pairing through the GCC anticodon of E. coli tRNA-Gly.

The temperature-jump method was used to compare the stability of anticodon-anticodon duplexes formed by the self-association of two tRNAs: yeast tRNA-Asp and Escherichia coli tRNA-Gly. Yeast tRNA-Asp duplexes contain a U/U mismatch while E. coli tRNA-Gly dimers have a C/C mismatch in the middle position of their quasi self-complementary anticodons GUC and GCC, respectively. At neutral pH, it is found that only tRNA-Asp duplexes exist whereas at pH 5.0 only tRNA-Gly duplexes are formed. This reflects the hemiprotonation of the N3 of the cytosines at pH 5.0 which induces a pairing between the two middle residues of the anticodon GCC in E. coli tRNA-Gly. This is the first evidence that a protonated C-C(+) base pair is compatible with the formation of a double helix with antiparallel strands in a natural RNA molecule.

Anticodon↗
PubMed1985

Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea.

Ethylnitrosourea is an alkylating reagent preferentially modifying phosphate groups in nucleic acids. It was used to monitor the tertiary structure, in solution, of yeast tRNAAsp and to determine those phosphate groups in contact with the cognate aspartyl-tRNA synthetase. Experiments involve 3' or 5'-end-labelled tRNA molecules, low yield modification of the free or complexed nucleic acid and specific splitting at the modified phosphate groups. The resulting end-labelled oligonucleotides are resolved on polyacrylamide sequencing gels and data analysed by autoradiography and densitometry. Experiments were conducted in parallel on yeast tRNAAsp and on tRNAPhe. In that way it was possible to compare the solution structure of two elongator tRNAs and to interpret the modification data using the known crystal structures of both tRNAs. Mapping of the phosphates in free tRNAAsp and tRNAPhe allowed the detection of differential reactivities for phosphates 8, 18, 19, 20, 22, 23, 24 and 49: phosphates 18, 19, 23, 24 and 49 are more reactive in tRNAAsp, while phosphates 8, 20 and 22 are more reactive in tRNAPhe. All other phosphates display similar reactivities in both tRNAs, in particular phosphate 60 in the T-loop, which is strongly protected. Most of these data are explained by the crystal structures of the tRNAs. Thermal transitions in tRNAAsp could be followed by chemical modifications of phosphates. Results indicate that the D-arm is more flexible than the T-loop. The phosphates in yeast tRNAAsp in contact with aspartyl-tRNA synthetase are essentially contained in three continuous stretches, including those at the corner of the amino acid accepting and D-arm, at the 5' side of the acceptor stem and in the variable loop. When represented in the three-dimensional structure of the tRNAAsp, it clearly appears that one side of the L-shaped tRNA molecule, that comprising the variable loop, is in contact with aspartyl-tRNA synthetase. In yeast tRNAPhe interacting with phenylalanyl-tRNA synthetase, the distribution of protected phosphates is different, although phosphates in the anticodon stem and variable loop are involved in both systems. With tRNAPhe, the data cannot be accommodated by the interaction model found for tRNAAsp, but they are consistent with the diagonal side model proposed by Rich & Schimmel (1977). The existence of different interaction schemes between tRNAs and aminoacyl-tRNA synthetases, correlated with the oligomeric structure of the enzyme, is proposed.

Alkylation↗
PubMed1985

Anticodon-anticodon interactions in solution. Studies of the self-association of yeast or Escherichia coli tRNAAsp and of their interactions with Escherichia coli tRNAVal.

The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNAAsp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNAAsp (GUC)-Escherichia coli tRNAVal (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNAAsp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant (Kass = 10(4)M-1 at 0 degrees C) is characteristic of a rather weak complex, when compared with that between tRNAAsp and tRNAVal (Kass = 4 X 10(6) M-1 at 0 degrees C), the effect being essentially linked to differences in the rate constant for dissociation. tRNAAsp split in the anticodon by T1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNAGly (GCC), E. coli tRNAVal (GAC) or E. coli tRNAAla (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNAAsp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNAAsp but its complex with E. coli tRNAVal was found to be only 1.5 times less stable than that between yeast tRNAAsp and E. coli tRNAVal. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNAAsp (in the presence of 1.6 M-ammonium sulphate and 3mM-spermine) revealed an important stabilization of the yeast and E. coli tRNAAsp duplexes or of their complexes with E. coli tRNAVal. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.

Anticodon↗
PubMed1985

Crystal structure of yeast tRNAAsp: atomic coordinates.

The atomic coordinates of yeast aspartic acid transfer RNA, as determined from a crystallographic investigation to 3 A resolution, are presented. In the ribose phosphate backbone sugars are in the C(3')-endo pucker, except for residues A7, A9, D16, G17, G18, D19, C20, U48, A58, and U60 which are in the C(2')-endo pucker. A least-squares superposition of the phosphorus atoms of yeast tRNAAsp and yeast tRNAPhe enlightens both an overall structural similarity and significant conformational differences. The largest deviations occur in the D-loop and the anticodon region.

Base Sequence↗
PubMed1985

The structure of yeast tRNA(Asp). A model for tRNA interacting with messenger RNA.

The anticodon of yeast tRNA(Asp), GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA(Asp) molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA(Phe). In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA(Asp) T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA(Phe). This variation is a consequence of the anticodon-anticodon base pairing which rigidifies the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA(Asp) substantiate such a correlation.

Anticodon↗
PubMed1984

Yeast tRNAAsp: codon and wobble codon-anticodon interactions. A transferred nuclear Overhauser enhancement study.

The conformations of the ribotrinucleoside bisphosphates GpApC and GpApU, the codon and wobble codon for aspartic acid respectively, bound to yeast tRNAAsp in solution, have been examined by means of time-dependent transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The conformations of the two bound ribotrinucleoside bisphosphates are shown to be very similar with an overall root-mean-square difference in interproton distances of 0.03 nm. The ribose conformations of all the residues are 3'-endo; the glycosidic bond torsion angles of the A and C residues of GpApC and of the A and U residues of GpApU are in the low anti range. These features are typical of an A-RNA type structure. In contrast, the G residue of both GpApC and GpApU exists as a mixture of syn and anti conformations. The overall conformation of the two bound ribotrinucleoside bisphosphates is also similar to A-RNA and the stability of the complexes is enhanced by extensive base-base stacking interactions. In addition, it is shown that the binding of the codon GpApC to tRNAAsp induces self-association into a multicomplex system consisting of four GpApC-tRNAAsp complexes, whereas the wobble codon GpApU fails to induce any observable self-association.

Anticodon↗
PubMed1984

Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases.

The interactions between beef tRNATrp with avian myeloblastosis reverse transcriptase have been studied by statistical chemical modifications of phosphate (ethylnitrosourea) and cytidine (dimethyl sulfate) residues, as well as by digestion of complexed tRNA by Cobra venom nuclease and Neurospora crassa endonuclease. Results with nucleases and chemicals show that reverse transcriptase interacts preferentially with the D arm, the anticodon stem and the T psi stem. All these regions are located in the outside of the L-shaped structure of tRNA. This domain of interaction is different to that reported previously in the complex of beef tRNA with the cognate aminoacyl-tRNA synthetase (M. Garret et al.; Eur. J. Biochem. In press). Avian reverse transcriptase destabilizes the region of tRNA where most of the tertiary interactions maintaining the structure of tRNA are located.

Alkylation↗
PubMed1984

Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase.

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl-tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and T psi arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable T psi stem. When alkylation by ethylnitrosourea was studied with the tRNATrp X tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase X tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.

Alkylation↗
PubMed1983

Large scale purification and structural properties of yeast aspartyl-tRNA synthetase.

A large scale purification procedure of baker's yeast aspartyl-tRNA synthetase is described which yields more than 200 mg pure protein starting from 30 Kg of wet commercial cells. The synthetase is an alpha 2 dimer of Mr = 125,000 +/- 5,000 which can be crystallized (J. Mol. Biol. 138, 1980, 129-135). The enzyme has an elongated shape with a Stokes radius of 50 A and a frictional ratio of 1.5. The synthetase has a tendency to aggregate but methods are described where this effect is overcome.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Structure of phenylalanine-accepting transfer ribonucleic acid and of its environment in aqueous solvents with different salts.

Thermodynamic and structural parameters were measured for brewers' yeast tRNAPhe in solution in the range of 0.1-0.9 M monovalent salt (with and without 1 mM MgCl2), pH 7.0, by small-angle neutron scattering. Partial specific volumes and preferential interaction parameters were found to be similar to corresponding values measured by more conventional means in DNA [Eisenberg, H. (1981) Q. Rev. Biophys. 14, 141-172]. There is no evidence of a large conformational change in tRNAPhe in this range, and the molecule has a radius of gyration that is the same as that calculated from the crystal-structure coordinates (23 A). Transfer RNA in solution is made up of polyion tRNA76- and 76 positive monovalent ions (in absence of Mg2+). The data show the polyion to be surrounded by a shell of solvent that is significantly denser than bulk, whose structure depends on salt conditions. In 0.1 M NaCl, it has an excess mass of approximately 85 molecules of water. This would be accounted for, for example, by approximately 850 molecules of water if their density were 10% higher than that for bulk. The radius of gyration of the dense shell is approximately 30 A for NatRNA and approximately 35 A for KtRNA. The present study shows that the solvent around tRNA is a component of its structure that must be taken into account in understanding its function.

Kinetics↗
PubMed1983

Crystallization of a tRNA . aminoacyl-tRNA synthetase complex. Characterization and first crystallographic data.

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Interaction of tRNAPhe and tRNAVal with aminoacyl-tRNA synthetases. A chemical modification study.

The alkylation by ethylnitrosourea of phosphodiester bonds in tRNAPhe from yeast and in tRNAVal from yeast and from rabbit liver and that by 4-(N-2-chloroethyl-N-methylamino)-benzylamine of N-7 atoms of guanosine residues in yeast tRNAVal have been used to study the interaction of these tRNAs with aminoacyl-tRNA synthetases. The modifications occurring at low yield were carried out on 3' and/or 5' end-labelled tRNAs either free or in the presence of cognate or non-cognate synthetases. After splitting of the tRNAs at the alkylated positions, the position of the modification sites in the tRNA sequences were detected by acrylamide gel electrophoresis. It was found that the synthetases protect against alkylation certain phosphate or guanosine residues in their cognate tRNAs. Non-cognate synthetases failed to protect efficiently specific positions in tRNA against modification. In yeast tRNAPhe the cognate phenylalanyl-tRNA synthetase protects certain phosphates located in all four stems and in the anticodon and extra-loop of the tRNA. Particularly strong protections occur on phosphate 34 in the anticodon loop and on phosphates 23, 27, 28, 41 and 46 in the D and anticodon stems. In yeast tRNAVal complexed with yeast valyl-tRNA synthetase the protected phosphates are essentially located in the corner between the amino-acid-accepting and D stems, in the D loop, anticodon stem and in the variable region of the tRNA. Three guanosine residues, located in the D stem, and another one in the 3' part of the anticodon stem were also found protected by the synthetase. In mammalian tRNAVal, complexed with the cognate but heterologous yeast valyl-tRNA synthetase, the protected phosphates lie in the anticodon stem, in the extra-loop and in the T psi arm. The location of the protected residues in the structure of three tRNAs suggests some common features in the binding of tRNAs to aminoacyl-tRNA synthetases. These results will be discussed in the light of informations on interaction sites obtained by nuclease digestion and ultraviolet cross-linking methods.

Alkylation↗
PubMed1983

Effect of elongation factor Tu on the conformation of phenylalanyl-tRNAPhe.

Structural features of the tRNAPhe molecule upon ternary complex formation with the bacterial elongation factor Tu were investigated. Phosphodiester bonds at positions 18 and 34 were found to be labilized in bound tRNA. Conversely, a higher stability of the phosphodiester links at positions 20, 21 and 36 was detected. Using ethylnitrosourea as a chemical probe a conformational change occurring at phosphate position 53 was observed in complexed tRNA. These results are interpreted by a structural rearrangement of the nucleic acid induced by complex formation.

Chemical Phenomena↗
PubMed1983

Yeast tRNAAsp-aspartyl-tRNA synthetase: the crystalline complex.

Aspartyl-tRNA synthetase from yeast, a dimer of molecular weight 125,000 and its cognate tRNA (Mr = 24,160) were co-crystallized using ammonium sulfate as precipitant agent. The presence in the crystals of both components in the two-to-one stoichiometric ratio was demonstrated by electrophoresis, biological activity assays and crystallographic data. Crystals belong to the cubic space group I432 with cell parameter of 354 A and one complex particle per asymmetric unit. The solvent content of about 78% is favorable for a low resolution structural investigation. By exchanging H2O for D2O in mother liquors, advantage can be taken from contrast variation techniques with neutron radiations. Diffraction data to 20 A resolution were measured at five different contrasts, two of them being close to the theoretical matching point of RNA and protein in the presence of ammonium sulfate. The experimental extinction of the diffracted signal was observed to be close to 36% D2O, significantly different from the predicted value of 41%. The phenomenon can be explained by the existence of a large interface region between the two tRNAs and the enzyme. These parts of the molecules are hidden from the solvent and their protons are less easily exchangeable. Accessibility studies toward chemicals of tRNAAsp in solution and in the presence of synthetase are in agreement with such a model.

Amino Acyl-tRNA Synthetases↗