Crystals of seryl-tRNA synthetase from Thermus thermophilus. Preliminary crystallographic data

Crystals have been obtained of seryl-tRNA synthetase from the extreme thermophile Thermus thermophilus, using mixed solutions of ammonium sulphate and methane pentane diol. The crystals are very stable and diffract to at least 2 A. The crystals are monoclinic (space group P21) with cell parameters a = 87.1 A, b = 126.9 A, c = 63.5 A and beta = 109.7 degrees.     

The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site.

The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution. The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits. The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem. In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs. This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA. Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition. These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.     

Comparative study of the reactability of phosphoric acid residues in tRNA(Ser) and tRNA(Leu) from Thermus thermophilus

The reactivity of phosphates in the Thermus thermophilus tRNA(Ser) (GCU) and tRNA(Leu) (CAG) was studied using the ethylnitrosourea modification. It was shown that phosphates of nucleotides 58-60 (T loop), 20-22 (D loop), and 48 (at the junction of the variable and T stems) were poorly modified in both tRNAs. The most pronounced differences in the reactivity were observed for phosphates at the junctions of the variable stem with T-stem (47q, 49) and anticodon stem (45). This indicates differences in orientations of the long variable arm relative to the backbone in the tRNAs studied.     

The contacts of yeast tRNA(Ser) with seryl-tRNA synthetase studied by footprinting experiments

Yeast tRNA(Ser) is a member of the class II tRNAs, whose characteristic is the presence of an extended variable loop. This additional structural feature raises questions about the recognition of these class II tRNAs by their cognate synthetase and the possibility of the involvement of the extra arm in the recognition process. A footprinting study of yeast tRNA(Ser) complexed with its cognate synthetase, yeast seryl-tRNA synthetase (an alpha 2 dimer), was undertaken. Chemical (ethylnitrosourea) and enzymatic (nucleases S1 and V1) probes were used in the experiments. A map of the contact points between the tRNA and the synthetase was established and results were analyzed with respect to a three-dimensional model of yeast tRNA(Ser). Regions in close vicinity with the synthetase are clustered on one face of tRNA. The extra arm, which is strongly protected from chemical modifications, appears as an essential part of the contact area. The anticodon triplet and a large part of the anticodon arm are, in contrast, still accessible to the probes when the complex is formed. These results are discussed in the context of the recognition of tRNAs in the aminoacylation reaction.     

Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.     

Phenylalanyl-tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNA(Phe).

Phenylalanyl-tRNA synthetase from the extreme thermophilic bacterium Thermus thermophilus can incorporate more than one molecule of phenylalanine into the tRNA(Phe). It is shown that the 'hyperaminoacylated' tRNA(Phe) is the bis-2',3'-O-phenylalanyl-tRNA(Phe), and its formation is typical for the thermophilic enzyme but does not occur for E. coli phenylalanyl-tRNA synthetase under the same conditions.     

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.     

tRNA(adenine-1-)-methyltransferase from Thermus thermophilus HB8

tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) was isolated from the extreme thermophile Thermus thermophilus strain HB8. The specific activity of the enzyme is about 50 000 and the yield of activity more than 20%. The method of isolation consists of five steps and is valid for isolation of mg quantities of the enzyme. The purified protein preparation is practically homogeneous in SDS-gel electrophoresis, the position of the protein band corresponds to a molecular weight of 25 000. By gel filtration on Sephadex G-100 the molecular weight of the native protein was found to be 70 000. These data allow to suggest a subunit structure of the enzyme. The enzyme is highly thermostable and is most active at 80 degrees C. The only activity of the enzyme is to methylate A58 in the T psi X loop of tRNA.     

Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA(Ser) acceptor stem by E.coli seryl-tRNA synthetase.

Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69. The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition. Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper. Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate. This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser). The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein. The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs.     

Peroxin Pex21p interacts with the C-terminal noncatalytic domain of yeast seryl-tRNA synthetase and forms a specific ternary complex with tRNA(Ser)

The seryl-tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis-related factor Pex21p. Several deletion mutants of seryl-tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two-hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C-terminal amino acids abolished Pex21p binding to seryl-tRNA synthetase. The catalytic parameters of purified truncated seryl-tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C-terminally appended domain of yeast seryl-tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA-binding motif, but it instead participates in the formation of a specific ternary complex with seryl-tRNA synthetase and tRNA(Ser), strengthening the interaction of seryl-tRNA synthetase with its cognate tRNA(Ser).     

Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution.

The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.     

Recognition mechanism of tRNA with tRNA(guanosine-2')methyltransferase from Thermus thermophilus HB 27

rRNA(Gm)methyltransferase from an extreme thermophile, Thermus thermophilus HB 27 specifically methylates the 2'-OH of the ribose ring of G18 in the invariant G18-G19 sequence in the D loop of tRNA. The interaction site on tRNA was presumed to be the D loop and stem structure. Destruction of tertiary structure of tRNA caused by heat resulted in a great decrease in the acceptor activity of methyl group. It was suggested by CD measurement that a conformational change of tRNA occurs when it forms an equimolar complex with Gm-methylase.     

Purification and characterization of tRNA(adenosine-1-)-methyltransferase from Thermus thermophilus HB27.

A58, the conserved adenosine residue in the T psi C loop of tRNAs, is methylated to m1A 58 in an extreme thermophile, Thermus thermophilus HB27. The enzyme catalyzing this methyltransfer reaction was purified from the thermophle. The substrate specificity of the enzyme was investigated by using tRNA fragments. The enzyme can transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, indicating that the main recognition site of the enzyme exists in the 3' half of tRNA including the T-loop and the T-stem.