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High-performance liquid chromatoraphy on an ion exchanger columm was successfully used for a rapid biochemical analysis of crystals of yeast tRNAAsp and aspartyl-tRNA synthetase as well as cocrystals formed by the synthetase and the tRNA.

A comparative study of the solution conformation of yeast tRNAAsp and tRNAPhe was undertaken with chemical reagents as structural probes. The accessibility of phosphates was assayed with ethylnitrosourea, that of N7 positions in guanines and adenines with dimethylsulfate and diethylpyrocarbonate and that of the N3 position in cytosines with dimethylsulfate. Statistical modifications were done on end-labelled tRNAs subsequently split at the modified positions and analyzed using sequencing gel methodologies. The results were correlated with the crystal conformations of both tRNAs which were analyzed with a graphic modelling program. Three particular positions are xiscussed. In all tRNAs so far studied, including tRNAAsp and tRNAPhe, phosphate 60 is protected against alkylation, even in the isolated 3’half-molecule of tRNAAsp. This indicates a common T-loop conformation in tRNA which is correlated with the constant presence of residue C61. A similar conformation is found in turnip yellow mosaic virus tRNA-like structure. Adenosine 21 and guanosine 45, two common residues in tRNAAsp and tRNAPhe, exhibit different reactivities against diethylpyrocarbonate and dimethylsulfate. A21 is reactive in tRNAAsp and protected in tRNAPhe; for G45 the situation is inversed. As to A21, the different reactivity is correlated with the extra-loop structures in the two tRNAs; in the case of G45 the results are explained by a different stacking of A9 between G45 and residue 46.

Chemical modification of tRNAs with base-specific reagents was successfully used for investigation of their structure in solution f 11. Using base-specific reagents allows the identi~cation of bases involved in hydrogen bond formation in tRNA structure. The development of a chemical modification method that would provide more information could be the use of reagents able to modify the riboses or the phosphates common to all nucleotide residues in tRNA. Studying the reactivity of these residues scattered throughout the entire tRNA molecule would then provide information about the backbone interactions in the tRNA and about the folding of the tRNA polynucleotide chain. We present here results of chemical modi~~t~on of yeast tRNAPhe with ethylnitrosourea, a reagent alkylating phosphodiester bonds in nucleic acids [2,3]. In the unfolded tRNAPhe all phosphates were reactive toward this reagent. In contrast in the folded tRNAPhe phosphates in different sites of the molecule were remarkably different in their reactivities toward the reagent. Phosphates in the exposed helical regions of tRNAphe(for instance in the anticodon arm) readily react with ethylnitrosourea, whilst the reactivity of some phosphates in the regions known to be involved in the tertiary structure formation is strongly reduced. The most pronouced effects were observed for the phosphates in positions 9,10,11,19,49,58,59 and 60.

The 3’-ends of several plant viral RNAs show a number of functional characteristics of tRNAs1,2; they are recognized by a set of tRNA-specific proteins, including aminoacyl-tRNA synthetases. So, the RNA of turnip yellow mosaic virus can be valylated by yeast valyl-tRNA synthetase3,4 with kinetic constants very close to those of the aminoacylation of yeast tRNAVal by this enzyme5. The 3′-ends of these viral RNAs however, are lacking several characteristic primary structural features of tRNAs such as strategic D- or T-loop sequences and modified bases. Moreover, they cannot be folded a priori into a canonical tRNA cloverleaf. In the case of TYMV RNA, the question arose as to “how do two structures as different as tRNAVal and the 3′-region of the viral RNA behave in such a similar fashion in the presence of valyl-tRNA synthetase?”. This question might be answered if one supposes that similar structural domains are recognized by the synthetase and exist at the level of the three dimensional structures and even at the level of their secondary structures. In this view, the secondary structure of the 3′-end of TYMV RNA was established by enzymatic footprinting methodologies6. A three-dimensional L-shaped conformation mimicking tRNA, but involving a new RNA folding principle, the pseudoknot, was proposed by the Leiden group7,8 for the 86 last nucleotides of this RNA. A rigourous graphical modelling allowed to assess the reality of this pseudoknotted folding9. To understand the involvement of the 3′-end of the TYMV RNA in aminoacylation, the direct contact points between this part of the RNA and yeast valyl-tRNA synthetase were determined10. Moreover, the study of the valylation of tRNA-like transcripts from cloned cDNA of TYMV RNA permitted to determine the minimal length of the RNA necessary for optimal valylation11.

A new way of studying RNA-protein complexes, using neutron small angle scattering in solution, is described and was applied in the case of the system, yeast valyl-tRNA synthetase, interacting with its cognate and non cognate yeast tRNAs. It was shown that, when limited amounts of tRNA (either cognate or non cognate) are added to valyl-tRNA synthetase, a complex consisting of two enzyme molecules and one tRNA molecule is first formed. It is subsequently dissociated to a one to one complex when more tRNA is present in the solution. The association curve shows a maximum for a molecular ratio, enzyme over tRNA, equal to 2.

The molecular structure of yeast tRNAAsp, a short extra-loop tRNA, was solved at 3 A resolution in two closely related crystal forms. The resulting model confirms the folding originally found in tRNAPhe: major differences concern the conformations of the loops and the relative positioning of the acceptor and anticodon stems which are more open, conferring to the tRNAAsp molecule its boomerang like shape. Crystal packing involves self-complementary GUC anticodon interactions thus making the structure a tempting model of a tRNA interacting with mRNA on the ribosome. The chemical stability of the tRNA in the crystalline state and in solution is compared using end-labelling and rapid sequencing gel methodologies. Partial splitting of the ribose-phosphate backbone in the anticodon loop was observed in solution but not in the crystal where the splitting is more pronounced in the D-loop.

Glycyl-tRNA synthetase from baker's yeast has been purified to homogeneity. This synthetase was found to be very sensitive to proteases present in the yeast extracts and to oxidizing agents of thiol groups. In the absence of protease inhibitors and/or dithioerythritol, the enzyme rapidly lost its activity and could not be isolated. The use of these protectors allowed us to obtain different oligomeric structures of the synthetase. In the presence of a minimal concentration of dithioerythritol but in the absence of protease inhibitors, a tetrameric glycyl-tRNA synthetase of the alpha 2 beta 2 type (alpha = 67 600, beta = 57 500) with a very low specific activity was recovered. With high concentrations of both protectors, a dimeric enzyme was isolated with a specific activity comparable to that for other yeast synthetases. The enzyme was of the alpha 2 type where alpha = 70 000--80 000 daltons, depending on whether phenylmethanesulfonyl fluoride or diisopropyl fluorophosphate was used as the protecting agent. The native form of the enzyme (alpha 2 = 160 000) associated easily with other proteins in various complexes of molecular weights from 250 000 to 300 000, some of them containing valyl-tRNA synthetase. The dimeric glycyl-tRNA synthetase was found in equilibrium with its subunits. Diluting the enzyme solution or increasing the salt concentration displaced the equilibrium toward the monomers, which are catalytically inactive for both the tRNA aminoacylation and the PPi-ATP exchange reactions. Addition of both tRNAGly and ATP.MgCl2 plus glycine displaced the equilibrium toward the dimeric form of the enzyme. Thiol groups were found to be involved in the association between the two subunits and in both activities of the synthetase. The results are interpreted in the light of possible regulatory mechanisms of the activity of this synthetase.

Yeast valyl-tRNA synthetase and its complexes with yeast tRNAVal were investigated by means of analytical ultracentrifugation. A molecular weight of 125 700 ± 1500 and a sedimentation coefficient (S020,w) of 6.3 ± 0.3 were found for the native enzyme. When the enzyme (3–60 μM) was mixed with its cognate tRNA, several types of complex were observed, depending on the relative amount of the two macromolecules. In the presence of equimolecular amounts of tRNA and enzyme, a complex formed by the association of one of each molecule was observed with a sedimentation coefficient of about 7.3 S. However, for tRNA enzyme stoichiometries lower than one, beside the 1 : 1 complex, a complex of higher molecular weight was observed, with a sedimentation coefficient of about 10.0 S which fits with the association of two valyl-tRNA synthetase molecules with one tRNA molecule. This 2 : 1 complex was predominant for tRNA enzyme stoichiometries lower than 0.3. It dissociated into the 1 : 1 complex upon addition of monovalent salts or MgCl2, suggesting the electrostatic nature of the interaction in this association. All these association and dissociation phenomena were detected over a large range of pH (6.0–7.5) and in various buffers.

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits of Mr, 64000 as shown by biochemical and crystallographic analyses. Previous studies have emphasized the high sensitivity of the amino-terminal region (residues 1–32) to proteolytic enzymes. This work reports the results of limited tryptic or chymotryptic digestion of the purified enzyme which gives rise to a truncated species that has lost the first 50–64 residues with full retention of both the activity and the dimeric structure. In contrast the larger tryptic fragment is distinguished from the whole enzyme by its weaker retention on heparin-substituted agarose gels. The cleaved N-terminal part presents peculiar structural features, such as a high content in lysine residues arranged in a palindromic fashion. The properties of the trypsin-modified enzyme and of the cleaved amino-terminal region are discussed in relation to the known structural characteristics of aspartyl-tRNA synthetase and of other eukaryotic aminoacyl-tRNA synthetases.

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 hemi- protonation 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.

The 20 aminoacyl-tRNAsynthetases, one specific for each amino acid, catalyse the following reaction: $$a{{a}_{1}} + ATP + tRN{{A}_{1}}\overset{{aa - tRNAsynthetase, M{{g}^{{ + + }}}}}{\rightleftharpoons} a{{a}_{1}} - tRN{{A}_{1}} + AMP + P{{P}_{i}}$$ In vivo these reactions are extremely specific and constitute a key step in the overall protein biosynthesis process.

Two forms of baker's yease valyl-tRNA synthetase have been purified to apparent homogeneity by classical methods. It was demonstrated that one of the two forms of the enzyme originates from the other by proteolysis, the respective amounts of each form depending on the physiological state of the yeast. The species mainly isolated from exponential growing yeast cells is a monomer of 130,000 daltons molecular weight. In stationary phase cells or in commercial yeast the major species is a degraded monomer of 120,000 daltons molecular weight ; however when the purification is carried out in the presence of phenylmethyl-sulphonyl fluoride, or diisopropylfluorophosphate large amounts of the not - degreded monomer can be obtained. Of great practical usefulness is the fact that large amounts of the native enzyme can be obtained pure after only two chromatographic steps on DEAE-cellulose and hydroxylapatite. The kinetic constants for valine, ATP and tRNAVal were determined, as well as the optimum aminoacylation conditions. It was found that the specific activity of the nondegraded valyl-tRNA synthetase is higher than that of the proteolysed enzyme for the aminoacylation reaction. On the contrary, both forms have the same ATP-pyroposphate exchange activity. The amino acids composition of the native enzyme was established. The tryptic fingerprints of the two valyl-tRNA synthetases were studied. Essentially similar maps were obtained. The number of the spots in the fingerprints indicates that the enzymes contain a high proportion of repeated sequences.

The interactions of yeast tRNAAsp with cognate aspartyl-tRNA synthetase have been studied in high concentrations of either sodium chloride or ammonium sulphate by fluorescence titration and small-angle neutron scattering. In solutions containing more than 1M NaCl no complex is formed and enzymatic activity is abolished. In strong contrast, however, the physical measurements showed the formation of a two-to-one tRNA-enzyme complex, with high affinity, in 1.6 M (NH4)2SO4. Aminoacylation assays under the same salt conditions showed the enzymatic fixation of aspartic acid to tRNAAsp to occur at an appreciable rate. The present study emphasizes that the effects of salts on protein-nucleic acid interactions do not depend only on ionic strength but also on the nature of the salt. This study has allowed a rational approach to the crystallisation of a functional tRNAAsp-aspartyl-tRNA synthetase complex (Giege, Lorber, Ebel, Thierry and Moras (1980) C.R. Acad. Sci. Paris, serie D, 291, 393-396).

Transfer ribonucleic acids (tRNAs) play a central role in the complex mechanism of protein synthesis. In that process their chief function is to carry amino acids to the ribosomes, to decode the messenger RNA and to incorporate the correct amino acid into the growing polypeptide chain.

In order to undertake structural and functional studies on the 3'-terminal part of turnip yellow mosaic virus RNA, a structure which can be specifically aminoacylated by valyl-tRNA synthetase, we have developed large-scale methods for purifying the tRNA-like sequence. Several experimental approaches were tested. One procedure was retained enabling us to purify large quantities of the homogeneous tRNA-like fragment. Starting from 1.5 g turnip yellow mosaic virus, one obtains 400 mg RNA, which is partially digested by T1 ribonuclease and which yields 1 - 2 mg pure tRNA-like fragment after three chromatographic steps: two filtrations on Ultrogel ACA 54 and onc revcrse-phase chromatography (RPC 5) in the presence of urea. A method has been worked out allowing preparation of 10 mg of the fragment per month. The purified RNA material appeared homogeneous upon polyacrylamide gel electrophoresis under denaturing conditions. The isolated tRNA-like structure can be valylated to an extent of 100% in the presence of purified yeast valyl-tRNA synthetasc with kinetic parameters resembling those of the tRNAVal aminoacylation.

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.

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.

Contacts between the tRNA-like domain in brome mosaic virus RNA and yeast tyrosyl-tRNA synthetase have been determined by footprinting with enzymatic probes. Regions in which the synthetase caused protections indicative of direct interaction coincide with loci identified by mutational studies as being important for efficient tyrosylation [Dreher, T. W. & Hall, T. C. (1988) J. Mol. Biol. 201, 41–55]. Additional extensive contacts were found upstream of the core of the tRNA-like structure. In parallel, the contacts of yeast tRNATyr with its cognate synthetase were determined by the same methodology and comparison of protected nucleotides in the two RNAs has permitted the assignment of structural analogies between domains in the viral tRNA-like structure and tRNATyr. Amino acid acceptor stems are similarly recognized by yeast tyrosyl-tRNA synthetase in the two RNAs, indicating that the pseudoknotted fold in the viral RNA does not perturb the interaction with the synthetase. A further important analogy appears between the anticodon/D arm of the L-conformation of tRNAs and a complex branched arm of the viral tRNA-like structure. However, no apparent anticodon triplet exists in the viral RNA. These results suggest that the major determinants for tyrosylation of yeast tRNATyr lie outside the anticodon stem and loop, possibly in the amino acid acceptor stem.

Different chemical reagents were used to study the tertiary structure of yeast tRNASer, a tRNA with a large variable region: ethylnitrosourea, which alkylates the phosphate groups; dimethylsulphate, which methylates N-7 of guanosine and N-3 of cytosine; and diethylpyrocarbonate, which modifies N-7 of adenine. The non-reactivity of N-3 of cytidine 47:1, 47:6, 47:7 and 47:8 and the reactivity of cytidine 47:3 confirms the existence of a variable stem of four base-pairs and a short variable loop of three residues. For the N-7 positions in purines, accessible residues are G1, G10, Gm18, G19, G30, I34, G35, A36, i6A37, G45, G47, G47:5, G47:9 and G73. The protection of N-7 atoms of residues G9, G15, A21, A22 and G47:9 reflects the tertiary folding. Strong phosphate protection was observed for P8 to P11, P20:1 to P22, P48 to P50 and for P59 and P60. A model was built on a PS300 graphic system on the basis of these data and its stereochemistry refined. While trying to keep most tertiary interactions, we adapted the tertiary folding of the known structures of tRNAAsp and tRNAPhe to the present sequence and solution data. The resulting model has the variable arm not far from the plane of the common L-shaped structure. A generalization of this model to other tRNAs with large variable regions is discussed.

Clones containing different lengths of cDNA corresponding to the 3' region of turnip yellow mosaic virus RNA were constructed and transcribed in vitro into the corresponding RNAs. Each transcript contained the L-shaped tRNA domain (N = 82) plus (i) in the case of 3 upstream sequences up to N = 93, 109 and 258; and (ii) in all cases an additional 6 nucleotide-stretch at the 5' end derived from the T7 promoter. The valylation of these molecules, as well as that of a fragment (N = 159) purified from viral RNA, was studied. Although all transcripts could be valylated by wheat germ valyl-tRNA synthetase, the 3 shorter fragments showed incomplete charging and slower rates, due mainly to lower Vmax values. Thus, although the tRNA-like L-shaped structure is the functional core permitting amino-acylation, upstream nucleotides between positions 82 and 159 play an important role in allowing the highest rates and levels of valylation. Structural arguments supporting this view are discussed.

The three-dimensional structure of two elongator tRNAs, tRNAPhe and tRNAAsp from yeast, has been solved at high resolution. Their secondary cloverleaf structures are folded in a tertiary L-shaped conformation with a more open structure for tRNAAsp. Both tRNAs present different molecular dynamics as revealed by temperature factors of the different parts of the molecules. These features are related to different conformational states of the tRNAs: tRNAPhe would possess the structure of a free tRNA and tRNAAsp that of a tRNA on the ribosome. The solution structure of both tRNAs was probed with chemical reagents and compared with the crystal structures. Structural similarities as well as differences were detected reflecting the versatility of tRNA structures.

The three-dimensional structures of two elongator transfer RNAs are known in great details: first that of yeast tRNA(Phe) (1–4) and more recently that of yeast tRNA(Asp) (5–8). As seen in Figure 1, both molecules are folded in an L-shaped conformation, which is also found for initiator tRNAs (9,10). Since the conserved or semi-conserved residues (11) are involved in the tertiary interactions which stabilize this folding (1–10), the L-shaped structure represents the general structural organization of all tRNA molecules. Such structural similarity is satisfactory to explain common functions of tRNAs, namely the interaction with ribosomes, and in the case of elongator tRNAs with elongator factors, but not sufficient to account for specific functions. In that case specific structural features must be involved. Differences in the anticodon sequences account for the decoding of the genetic message on mRNA. Recognition by aminoacyl-tRNA synthetases, which leads to the specific ami noacylation of tRNAs is more complex. Because many aminoacyl-tRNA synthetases recognize isoacceptor tRNAs and catalyze tRNA mischarging (12), these features cannot be simple linear nucleotide sequences, but more likely structural domains found at the three-dimensional level.

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.

Yeast aspartyl-tRNA synthetase is a dimeric enzyme (alpha 2, Mr 125,000) which can be crystallized either alone or complexed with tRNAAsp. When analyzed by electrophoretic methods, the pure enzyme presents structural heterogeneities even when recovered from crystals. Up to three enzyme populations could be identified by polyacrylamide gel electrophoresis and more than ten by isoelectric focusing. They have similar molecular masses and mainly differ in their charge. All are fully active. This microheterogeneity is also revealed by ion-exchange chromatography and chromatofocusing. Several levels of heterogeneity have been defined. A first type, which is reversible, is linked to redox effects and/or to conformational states of the protein. A second one, revealed by immunological methods, is generated by partial and differential proteolysis occurring during enzyme purification from yeast cells harvested in growth phase. As demonstrated by end-group analysis, the fragmentation concerns exclusively the N-terminal end of the enzyme. The main cleavage points are Gln-19, Val-20 and Gly-26. Six minor cuts are observed between positions 14 and 33. The present data are discussed in the perspective of the crystallographic studies on aspartyl-tRNA synthetase.

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.

The conformation of the T-loop of yeast tRNA(Asp) was studied by structural mapping techniques using chemical and enzymatic probes and by three-dimensional graphics modeling with the known crystallographic structures of tRNAs as references. The structural importance of C61 (conserved in the T-stem of all tRNAs) for the loop conformation was directly checked by ethylnitrosourea phosphate alkylation, either on the 3'-half tRNAAsp molecule or on a variant in which C61 was replaced by U61. The reactivity of P60 against ethylnitrosourea alkylation in the variant emphasizes the role of the hydrogen bond between this phosphate and position N4 of C61 for stabilizing the conformation of the T-loop. Experiments on several tRNA variants, containing C61 but altered in the sequence or in the length of the T-loop, indicate that other structural features help to stabilize the hydrogen bond network around P60. Evidence is presented that the reverse Hoogsteen base pair T54-A58 contributes to this stabilization by maintaining the hydrogen bonding between the 2'OH of ribose 58 and P60. Using graphics modeling and based on the chemical data. T-loops of several variants were constructed. It appears that both the constant length of the T-loop and the presence of psi 55 are crucial for the correct interaction between the T- and D-loops. The conclusion of this study is that the T-loop in tRNA possesses an intrinsic conformation (mainly governed by the constant residues) existing primarily without the structural context of the entire tRNA molecule.

Systematic crystallization studies on brewer's yeast aspartic acid transfer RNA have yielded different crystal forms, one of them diffracting to 3 A resolution. The high resolution crystal form is orthorhombic ( C222 1 , a = 61 A , b = 68 A , c = 148 A with one molecule per asymmetric unit) and is stable for over four days under X-rays.

Purification and crystallization of macromolecules are often challenging steps in structural projects using X-ray diffraction methods (1,2) and in some cases they can represent limiting factors. This explains the recent interest of molecular biologists and physicists to better understand crystal growth of macromolecules (3). In what follows we will discuss the role of purification in the crystallization of biopolymers and give particular emphasis to the concept of purity of preparations. Experimental set ups and properties of precipitants used to induce crystallization will be reviewed. Particular parameters affecting crystal growth of proteins (i.e. proteolysis) or of nucleic acids (i.e. nature of counter-ions, nuclease digestion, chemical fragility of RNAs) will be discussed. Examples taken from our laboratory in the field of aminoacyl-tRNA synthetases and transfer ribonucleic acids, and more generally in the field of nucleoproteins will illustrate the subject.

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.

IgG purified from sera of several patients with systemic lupus erythematosus and hepatitis B are shown to present RNA hydrolyzing activities that are different from the weak RNase A-type activities found in the sera of healthy donors. Further investigation brings evidence for two intrinsic activities, one observed in low salt conditions and another specifically stimulated by Mg2+ions and distinguishable from human sera RNases. Cleavage of RNA substrates by the latter activity is not sequence-specific but sensitive to both subtle conformational and/or drastic folding changes, as evidenced by comparative analysis of couples of structurally well-studied RNA substrates. These include yeast tRNAAsp and its in vitro transcript and human mitochondrial tRNALys-derived in vitro transcripts. The discovery of catalytic antibodies with RNase activities is a first step towards creation of a new generation of tools for the investigation of RNA structure.

Three new crystal forms of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase have been produced. The best crystals, obtained after modifying both purification and crystallization conditions, belong to space group P212121 and diffract to 2.7 A. Unit cell parameters are a = 210.4 A , b = 145.3 A and c = 86.0 A (1 A = 0.1 nm), with one dimeric enzyme and two tRNA molecules in the asymmetric unit.

Glutaminyl-tRNA synthetase from Deinococcus radiodurans (GlnRS-Dr) distinguishes from known GlnRSs by the presence additional long insertions. A C-terminal extension (C-ter) is predicted to adopt the same fold as part of an amidotransferase (AdT) subunit making this GlnRS almost unique among its homologs since it corresponds to the fusion of protein domains from both direct and indirect pathways of tRNA aminoacylation. In the crystal structure of GlnRS-Dr, C-ter was not solved although the unit cell was large enough to accommodate it. In the same time, NMR analysis of the extension confirmed the presence of a well structured region. Additional structures were solved in the presence of small substrates (glutamine, 2 adenylate analogs) and the C-terminal truncated form as well. In 2 cases, it leads to the stabilization of an insertion loop closing the active site. Structure analysis, functional implication, crystal packing and plasticity properties are discussed., La GlnRS de Deinococcus radiodurans se distingue des autres GlnRS par la présence d'un appendice additionnel en C-terminal (C-ter). Celui-ci adopterait le même repliement que la famille de protéines YqeY de fonction inconnue et une région de la sous-unité GatB de l'AdT. Son architecture atypique, trouvée dans 4 organismes, corresponds à la fusion de protéine de la voie directe et indirecte d'aminoacylation des ARNt. La structure cristallographique de la GlnRS-Dr n'a pas permis de résoudre la région C-ter, la maille étant suffisamment large pour l'accommoder. Des analyses en RMN du C-ter isolé ont confirmé la présence d'une région majoritairement structurée. D'autres structures ont été résolues en présence de petits substrats (glutamine, analogues d'adénylate) ainsi que la forme tronquée en C-ter. Dans 2 cas, une conformation verrouillée unique du site actif a été mise en évidence. Des analyses structurales et fonctionnelles et les propriétés de l'empilement cristallin sont exposées.

My thesis focuses on the functional and structural characterization of the tyrosine specific aminoacylation system from human mitochondria. Unlike the general situation found in other aminoacylation systems, cross-reactions between bacteria and archea/eukarya are impossible due to the difference of tRNATyr first base pair. Human mitochondrial tyrosyl-tRNA synthetase (TyrRS) is the first known TyrRS overcoming the species barrier and that doesn't discriminate tRNATyr for their first base pair. The mitochondrial TyrRS is an homodimer of elongated shape that will like likely bind one tRNATyr molecule across both subunits. It differs from other TyrRS by the presence of two insertions at its surface, the first one being potentially implicated in the tRNATyr binding and the second one that could serve as a co-factor binding interface., Ma thèse porte sur l'étude fonctionnelle et structurale des partenaires de la réaction d'aminoacylation spécifique de la tyrosine dans la mitochondrie humaine. Contrairement à ce qui a été observé pour les autres systèmes d'aminoacylation, les réactions de charges croisées entre bactéries et archaea/eucaryotes sont impossibles suite à la nature différente de la première paire de bases de l'ARNtTyr. La tyrosyl-ARNt synthétase (TyrRS) mitochondriale humaine est la première TyrRS connue à ce jour qui s'affranchisse de la barrière d'espèces et qui ne discrimine pas les ARNtTyr en fonction de la nature de leur première paire de bases. La TyrRS mitochondriale est un homodimère de forme allongée susceptible de fixer l'ARNtTyr à cheval sur ses deux monomères. Elle se distingue des autres TyrRS par la présence de deux insertions à sa surface, l'une potentiellement impliquée dans la reconnaissance de l'ARNtTyr et l'autre qui pourrait constituer une zone d'interaction avec un cofacteur.

Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases (aaRS), is essential to insure translational fidelity. Here, it is shown that expression of AspRS is regulated in Saccharomyces cerevisiae by a feedback mechanism, that necessitates the binding of AspRS to its messenger RNA. The correlation between AspRS expression and mRNAAspRS and tRNAAsp concentrations, as well as the presence of AspRS in the nucleus, suggest an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNAAsp, is imported in the nucleus where it binds to mRNAAspRS and thus inhibits its accumulation. We have established the folding of the 300-nucleotides long 5' end of mRNAApRS and identified the structural signals involved in the regulation process. We propose that the mRNAAspRS fragment folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNAAsp anticodon-like stem-loop structure that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, support a model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure like it recognizes its cognate tRNAAsp.Finally, the consequences of an increased concentration of AspRS in the cell have been tested. In vitro, high AspRS concentrations lead to mis-aspartylation of tRNAAsn and tRNAGlu. In vivo, the design of a reporter gene conferring an antibiotic resistance, dependent on mischarged tRNAs, did not allow to detect any cross aminoacylation. However, the proteomic analysis of yeasts overexpressing AspRS pointed out the conditions of AspRS accumulation in the cell by detecting the presence of an additional control mechanism at the post-translational level.