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.     

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.     

Nuclear Overhauser effect study and assignment of D stem and reverse-Hoogsteen base pair proton resonances in yeast tRNAAsp

Nuclear Overhauser effects (NOEs) in yeast tRNAAsp were found for all four GU and G psi base pairs. NOEs of both reverse-Hoogsteen pairs were identified by comparison with a purine C8 deuterated sample. Several NOEs involving these resonances were also found which are clearly between single protons on adjacent base pairs. These interbase NOEs, combined with the assumption of reasonable similarity between the structure of yeast tRNAAsp and that of yeast tRNAPhe, lead to unambiguous assignment of many resonances including all the ring NH and C2 protons in the D stem. The stability of the stem at 28 degrees C, as recently deduced by Moras et al (Nature 288 669-674), from x-ray diffraction is confirmed. Assignments of the ring NH resonances of T54-A58 and of a G psi pair are made for the first time.     

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.     

Proton nuclear magnetic resonance of minor nucleosides in yeast phenylalanine transfer ribonucleic acid. Conformational changes as a consequence of aminoacylation, removal of the Y base, and codon--anticodon interaction.

The assignments of the resonances of the methyl and methylene groups belonging to the residues dihydro-uridine-16 and -17 (C5 and C6), dimethylguanosine-26, N-2-methylguanosine-10, and 7-methylguanosine-46 of yeast tRNAPhe at low temperature are reported. Observing the high-field proton NMR spectral region at different temperatures, the effects of aminoacylation, removal of the Y base, and codon-anticodon interaction on the tertiary structure of yeast tRNAPhe were investigated. The following are the results of this study. (1) The two dihydrouridine residues of tRNAPhe have different environments in aqueous solution: dihydro-uridine-16 is more shielded than dihydrouridine-17. (2) The ribothymidine residue from the fragment (47--76) of yeast tRNAPhe and from a tRNA with a partially disrupted structure exhibits multiple conformations arising from different stacking modes between the ribothymidine-54 and the guanosine-53 residue. (3) Upon aminoacylation the type of guanosine-53 interaction with ribothymidine-54 in the tRNAPhe changes. (4) Removal of the Y base from the anticodon loop of yeast tRNAPhe weakens the thermal stability of the tertiary interactions. (5) The interaction of two complementary anticodons in the absence of proteins and of ribosomes results in stabilization of the tertiary structure. Codon-anticodon interaction dependent rearrangement of the tertiary structure of yeast tRNAPhe was not observed. The spin-lattice relaxation times of the methyl and methylene groups of the minor nucleosides in yeast tRNAPhe demonstrate that the minor nucleosides undergo rotational reorientation (tau c) in the nano-second range. The observed differences in these tau c values indicate a similarity of structure of tRNAPhe in solution and in crystalline form.     

Structure of the ribotrinucleoside diphosphate codon UpUpC bound to tRNAPhe from yeast. A time-dependent transferred nuclear Overhauser enhancement study

The structure of the ribotrinucleoside diphosphate UpUpC, the codon for phenylalanine, bound to yeast tRNAPhe in solution is elucidated using time-dependent proton-proton transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The glycosidic bond and ribose conformations are low anti and 3'-endo, respectively, typical of an A-RNA type structure. The main chain torsion angles are all within the range of those expected for A-RNA but small differences from those in conventional A-RNA 11 result in a special structure with a larger rotation per residue (40 to 45 degrees compared to 32.7 degrees in R-RNA 11) and almost perfect stacking of the bases. These two structural features, which are similar to those found in the anticodon triplet of the monoclinic crystal form of tRNAPhe, can account for the known greater stability of the codon-anticodon complex relative to an equivalent double helical RNA trimer with a conventional A-RNA structure.     

Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.     

Effect of excision of the Y-base on the interaction of tRNAPhe (yeast) with phenylalanyl-tRNA synthetase (yeast).

The interaction between tRNAPhe (yeast), from which the Y-base has been removed by acid treatment, and phenylalanyl-tRNA synthetase (yeast) has been investigated by fluorescence competition titrations and sedimentation velocity runs. The binding parameters are given under various ionic conditions. The tRNAPhe-Y still can occupy the specific binding sites on the enzyme. Compared to unmodified tRNAPhe, the binding constant is lowered by more than one order of magnitude. It can be concluded that the Y-base is not necessary for specific recognition of tRNAPhe by the cognate synthetase, it rather may represent a point of attachment for the synthetase.     

In vitro construction of yeast tRNAAsp variants: nucleotide substitutions and additions in T-stem and T-loop.

A procedure for the construction of 3'-end labelled yeast tRNAAsp harboring substitutions or additions of any desired nucleotide in T-stem and T-loop (position 57 to 61) has been developed. This was done by in vitro enzymatic manipulations of the yeast tRNAAsp involving specific hydrolysis with RNases, phosphorylation and dephosphorylation with T4 polynucleotide kinase and ligation with T4 RNA ligase. Using this procedure we have replaced conserved or semi-conserved nucleotides located in position 57 to 61 of yeast tRNAAsp. We have also constructed different yeast tRNAAsp with eight bases instead of seven in T-loop. Further use of these tRNAAsp variants will be discussed with the help of the crystallographic three-dimensional structure.     

Solution structure of a tRNA with a large variable region: yeast tRNASer

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.     

Evidence for the existence of an expressed minor variant tRNAPhe in yeast

Two expressed brewer's yeast tRNAsPhe, a major and a minor one, have been purified and sequenced. The major tRNAPhe corresponds to the already known tRNAPhe, whereas the minor one differs from the former in the substitution of T6-A67 by C6-G67 base pair in the "acceptor stem". The minor tRNAPhe contaminates all preparations of yeast tRNAPhe except those prepared by polyacrylamide gel electrophoresis.     

Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.     

A high resolution diffracting crystal form of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase

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 P2(1)2(1)2(1) 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.     

The solution structure of a RNA pentadecamer comprising the anticodon loop and stem of yeast tRNAPhe. A 500 MHz 1H-n.m.r. study.

A 500 MHz 1H-n.m.r. study on the semi-synthetic RNA pentadecamer 5'-r(C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G) comprising the anticodon loop and stem (residues 28-42) of yeast tRNAPhe is presented. By using pre-steady-state nuclear-Overhauser-effect measurements all exchangeable and non-exchangeable base proton resonances, all H1' ribose resonances and all methyl proton resonances are assigned and over 70 intra- and inter-nucleotide interproton distances determined. From the distance data the solution structure of the pentadecamer is solved by model-building. It is shown that the pentadecamer adopts a hairpin-loop structure in solution with the loop in a 3'-stacked conformation. This structure is both qualitatively and quantitatively remarkably similar to that of the anticodon loop and stem found in the crystal structures of tRNAPhe with an overall root-mean-square difference of 0.12 nm between the interproton distances determined by n.m.r. and X-ray crystallography. The hairpin-loop solution structure of the pentadecamer is very stable with a 'melting' temperature of 53 degrees C in 500 mM-KCl, and the structural features responsible for this high stability are discussed. Interaction of the pentadecamer with the ribotrinucleoside diphosphate UpUpC, one of the codons for the amino acid phenylalanine, results only in minor perturbations in the structure of the pentadecamer, and the 3'-stacked conformation of the loop is preserved. The stability of the pentadecamer-UpUpC complex (K approximately 2.5 X 10(4) M-1 at 0 degrees C) is approximately an order of magnitude greater than that of the tRNAPhe-UpUpC complex.     

Specificity and mechanism of the cleavages induced in yeast tRNAPhe by magnesium ions

The specificity of magnesium ion-induced hydrolysis of yeast tRNAPhe in solution was studied as a function of the excess of Mg(II) ions and pH. The major cuts at phosphates 16 and 20 as well as minor cleavages at phosphates 17, 18, 21, 34 and 36 occur at all pH values in the range of 8.0-9.5, and at a molar excess of magnesium ions over the tRNA ranging from 125 to 5000. In yeast tRNA(Phe)-Y the efficiency of the anticodon and D-loop cleavages is considerably decreased while the differently modified Y-base of yellow lupin tRNA(Phe) lowers the specificity of the weak anticodon loop cleavages. The mechanism of the Mg(II)-induced cleavages is discussed on the basis of yeast tRNA(Phe) crystal structure data, and the two major D-loop cleavages are thought to be effected from two distinct magnesium binding sites. The possibility of probing the environments of magnesium binding sites in tRNAs by the induced cleavages is demonstrated, and the relevance of magnesium-induced tRNA cleavages to RNA catalysis is discussed.     

Enzymatic conversion of guanosine 3'' adjacent to the anticodon of yeast tRNAPhe to N1-methylguanosine and the wye nucleoside: dependence on the anticodon sequence.

N1-Methylguanosine (m1G) or wye nucleoside (Y) are found 3' adjacent to the anticodon (position 37) of eukaryotic tRNAPhe. The biosynthesis of these two modified nucleosides has been investigated. The importance of the type of nucleosides in the anticodon of yeast tRNAPhe on the potentiality of this tRNA to be a substrate for the corresponding maturation enzyme has also been studied. This involved microinjection into Xenopus laevis oocytes and incubation in a yeast extract of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent Y nucleoside were substituted by various tetranucleotides ending with a guanosine. The results obtained by oocyte microinjection indicate: that all the restructured yeast tRNAsPhe are efficient substrates for the tRNA (guanosine-37 N1)methyltransferase. This means that the anticodon sequence is not critical for the tRNA recognition by this enzyme; in contrast, for Y nucleoside biosynthesis, the anticodon sequence GAA is an absolute requirement; the conversion of G-37 into Y-37 nucleoside is a multienzymatic process in which m1G-37 is the first obligatory intermediate; all the corresponding enzymes are cytoplasmic. In a crude yeast extract, restructured yeast tRNAPhe with G-37 is efficiently modified only into m1G-37; the corresponding enzyme is a S-adenosyl-L-methionine-dependent tRNA methyltransferase. The pure Escherichia coli tRNA (guanosine-37 N1) methyltransferase is unable to modify the guanosine-37 of yeast tRNAPhe.     

Influence of various factors on the recognition specificity of tRNAs by yeast valyl-tRNA synthetase.

Using filtration through nitrocellulose membranes we found that complexes between yeast valyl-tRNA synthetase can easily be detected at low pH and ionic strength with the cognate tRNAVal, but also with several non-cognate tRNAs (tRNAPhe, tRNATyr, tRNAMet and tRNAAsp). We show here that the amino acid linked to the tRNA has no detectable effect on these interactions. The influence of various factors on the discrimination by the enzyme between the cognate and the non-cognate tRNAs has been studied. An increase in pH or ionic strength leads to a decrease in the same ratio of the affinity constants between the enzyme and the cognate as well as the noncognate tRNA. The addition of organic solvents has little effect on these constant either in the cognate or in the non-cognate systems; the addition of substrates of the aminoacylation reaction has not effect on the ratio between the constants. This similar behaviour suggests that at least part of the specific of non-specific interactions must be identical. On the contrary, magnesium between 1 mM and 50 mM increases the specificity of recognition, showing the importance of slight conformational changes in the tRNA molecule to the specificity of interaction.     

Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid.

The low-field hydrogen-bond ring NH proton nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acids (tRNAs) related to yeast tRNAPhe have been examined in detail. Several resonances are sensitive to magnesium ion and temperature, suggesting that they are derived from tertiary base pairs. These same resonances cannot be attributed to cloverleaf base pairs as shown by experimental assignment and ring current shift calculation of the secondary base pair resonances. The crystal structure of yeast tRNAPhe reveals at least six tertiary base pairs involving ring NH hydrogen bonds, which we conclude are responsible for the extra resonances observed in the low-field NMR spectrum. In several tRNAs with the same tertiary folding potential and dihydrouridine helix sequence as yeast tRNAPhe, the extra resonances from tertiary base pairs are observed at the same position in the spectrum.     

Unusual structures in single-stranded ribonucleic acid: proton nuclear magnetic resonance of AUCCA in deuterium oxide

Conformational features of the oligoribonucleic acid (oligo-RNA) A1-U2-C3-C4-A5 are explored by proton nuclear magnetic resonance (NMR). The sequence is a molecular cognate of a portion of the T psi C loop and stem regions of yeast tRNAPhe. The molecule forms at least two classes of flexible yet ordered structures. Class I states are similar in spectral properties to the component oligomers, AU, AUC, and AUCC, and are likely to be standard right-helical structures. Class II states are characterized by a 2'-endo pucker at A1 and unusually large shielding of several C3 and U2 protons. Most of these features are consistent with identifying the class II solution structures with the "arch" conformation for the T psi C region determined by X-ray crystallography of yeast tRNAPhe.     

Transcription and processing of intervening sequences in yeast tRNA genes.

Genes for yeast tRNATyr and tRNAPhe have been sequenced (Goodman, Olson and Hall, 1977; Valenzuela et al., 1978) which contain additional nucleotides (intervening sequences) within the middle of the gene that are not present in the mature tRNA. We have isolated precursors to rRNATyr and tRNAPhe from a yeast temperature-sensitive mutant (at the rna1 locus) which accumulates only certain precursor tRNAs at the nonpermissive temperature. The tRNATyr and tRNAPhe precursors were analyzed by oligonucleotide mapping; they each contain the intervening sequence and fully matured 5' and 3' termini. Furthermore, these precursors were used as substrates to search for an enzymatic activity which can remove the intervening sequences and religate the ends. We have shown that wild-type yeast contains such an activity, and that this activity specifically removes the intervening sequences to produce mature-sized RNAs.