Purification, crystallization and preliminary X-ray crystallographic analysis of mammalian translation elongation factor eEF1A2.

Translation elongation factor eEF1A2 was purified to homogeneity from rabbit muscle by two consecutive ion-exchange column-chromatography steps and this mammalian eEF1A2 was successfully crystallized for the first time. Protein crystals obtained using ammonium sulfate as precipitant diffracted to 2.5 Å resolution and belonged to space group P6(1)22 or P6(3)22 (unit-cell parameters a = b = 135.4, c = 304.6 Å). A complete native data set was collected to 2.7 Å resolution.

The 2.9 A crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA(Ser).

The crystal structure of Thermus thermophilus seryl-transfer RNA synthetase, a class 2 aminoacyl-tRNA synthetase, complexed with a single tRNA(Ser) molecule was solved at 2.9 A resolution. The structure revealed how insertion of conserved base G20b from the D loop into the core of the tRNA determines the orientation of the long variable arm, which is a characteristic feature of most serine specific tRNAs. On tRNA binding, the antiparallel coiled-coil domain of one subunit of the synthetase makes contacts with the variable arm and T psi C loop of the tRNA and directs the acceptor stem of the tRNA into the active site of the other subunit. Specificity depends principally on recognition of the shape of tRNA(Ser) through backbone contacts and secondarily on sequence specific interactions.

Crystal structures at 2.5 angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate.

Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 A resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine side chain, the interactions are likely to be similar in all class 2 synthetases.

Molecular modeling and molecular dynamics simulation study of archaeal leucyl-tRNA synthetase in complex with different mischarged tRNA in editing conformation.

Aminoacyl-tRNA synthetases (aaRSs) play important roles in maintaining the accuracy of protein synthesis. Some aaRSs accomplish this via editing mechanisms, among which leucyl-tRNA synthetase (LeuRS) edits non-cognate amino acid norvaline mainly by post-transfer editing. However, the molecular basis for this pathway for eukaryotic and archaeal LeuRS remain unclear. In this study, a complex of archaeal P. horikoshii LeuRS (PhLeuRS) with misacylated tRNALeu was modeled wherever tRNA's acceptor stem was oriented directly into the editing site. To understand the distinctive features of organization we reconstructed a complex of PhLeuRS with tRNA and visualize post-transfer editing interactions mode by performing molecular dynamics (MD) simulation studies. To study molecular basis for substrate selectivity by PhLeuRS's editing site we utilized MD simulation of the entire LeuRS complexes using a diverse charged form of tRNAs, namely norvalyl-tRNALeu and isoleucyl-tRNALeu. In general, the editing site organization of LeuRS from P.horikoshii has much in common with bacterial LeuRS. The MD simulation results revealed that the post-transfer editing substrate norvalyl-A76, binds more strongly than isoleucyl-A76. Moreover, the branched side chain of isoleucine prevents water molecules from being closer and hence the hydrolysis reaction slows significantly. To investigate a possible mechanism of the post-transfer editing reaction, by PhLeuRS we have determined that two water molecules (the attacking and assisting water molecules) are localized near the carbonyl group of the amino acid to be cleaved off. These water molecules approach the substrate from the opposite side to that observed for Thermus thermophilus LeuRS (TtLeuRS). Based on the results obtained, it was suggested that the post-transfer editing mechanism of PhLeuRS differs from that of prokaryotic TtLeuRS.

tRNA(Pro) anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase.

BACKGROUND: Most aminoacyl-tRNA synthetases (aaRSs) specifically recognize all or part of the anticodon triplet of nucleotides of their cognate tRNAs. Class IIa and class IIb aaRSs possess structurally distinct tRNA anticodon-binding domains. The class IIb enzymes (LysRS, AspRS and AsnRS) have an N-terminal beta-barrel domain (OB-fold); the interactions of this domain with the anticodon stem-loop are structurally well characterised for AspRS and LysRS. Four out of five class IIa enzymes (ProRS, ThrRS, HisRS and GlyRS, but not SerRS) have a C-terminal anticodon-binding domain with an alpha/beta fold, not yet found in any other protein. The mode of RNA binding by this domain is hitherto unknown as is the rationale, if any, behind classification of anticodon-binding domains for different aaRSs. RESULTS: The crystal structure of Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) in complex with tRNA(Pro) has been determined at 3.5 A resolution by molecular replacement using the native enzyme structure. One tRNA molecule, of which only the lower two-thirds is well ordered, is found bound to the synthetase dimer. The C-terminal anticodon-binding domain binds to the anticodon stem-loop from the major groove side. Binding to tRNA by ProRSTT is reminiscent of the interaction of class IIb enzymes with cognate tRNAs, but only three of the anticodon-loop bases become splayed out (bases 35-37) rather than five (bases 33-37) in the case of class IIb enzymes. The two anticodon bases conserved in all tRNA(Pro), G35 and G36, are specifically recognised by ProRSTT. CONCLUSIONS: For the synthetases possessing the class IIa anticodon-binding domain (ProRS, ThrRS and GlyRS, with the exception of HisRS), the two anticodon bases 35 and 36 are sufficient to uniquely identify the cognate tRNA (GG for proline, GU for threonine, CC for glycine), because these amino acids occupy full codon groups. The structure of ProRSTT in complex with its cognate tRNA shows that these two bases specifically interact with the enzyme, whereas base 34, which can be any base, is stacked under base 33 and makes no interactions with the synthetase. This is in agreement with biochemical experiments which identify bases 35 and 36 as major tRNA identity elements. In contrast, class IIb synthetases (AspRS, AsnRS and LysRS) have a distinct anticodon-binding domain that specifically recognises all three anticodon bases. This again correlates with the requirements of the genetic code for cognate tRNA identification, as the class IIb amino acids occupy half codon groups.

The structural basis for seryl-adenylate and Ap4A synthesis by seryl-tRNA synthetase.

BACKGROUND: Seryl-tRNA synthetase is a homodimeric class II aminoacyl-tRNA synthetase that specifically charges cognate tRNAs with serine. In the first step of this two-step reaction, Mg.ATP and serine react to form the activated intermediate, seryl-adenylate. The serine is subsequently transferred to the 3'-end of the tRNA. In common with most other aminoacyl-tRNA synthetases, seryl-tRNA synthetase is capable of synthesizing diadenosine tetraphosphate (Ap4A) from the enzyme-bound adenylate intermediate and a second molecule of ATP. Understanding the structural basis for the substrate specificity and the catalytic mechanism of aminoacyl-tRNA synthetases is of considerable general interest because of the fundamental importance of these enzymes to protein biosynthesis in all living cells. RESULTS: Crystal structures of three complexes of seryl-tRNA synthetase from Thermus thermophilus are described. The first complex is of the enzyme with ATP and Mn2+. The ATP is found in an unusual bent conformation, stabilized by interactions with conserved arginines and three manganese ions. The second complex contains seryl-adenylate in the active site, enzymatically produced in the crystal after soaking with ATP, serine and Mn2+. The third complex is between the enzyme, Ap4A and Mn2+. All three structures exhibit a common Mn2+ site in which the cation is coordinated by two active-site residues in addition to the alpha-phosphate group from the bound ligands. CONCLUSIONS: Superposition of these structures allows a common reaction mechanism for seryl-adenylate and Ap4A formation to be proposed. The bent conformation of the ATP and the position of the serine are consistent with nucleophilic attack of the serine carboxyl group on the alpha-phosphate by an in-line displacement mechanism leading to the release of the inorganic pyrophosphate. A second ATP molecule can bind with its gamma-phosphate group in the same position as the beta-phosphate of the original ATP. This can attack the seryl-adenylate with the formation of Ap4A by an identical in-line mechanism in the reverse direction. The divalent cation is essential for both reactions and may be directly involved in stabilizing the transition state.

A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: comparison with histidyl-tRNA synthetase.

We describe the recognition by Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) of proline, ATP and prolyl-adenylate and the sequential conformational changes occurring when the substrates bind and the activated intermediate is formed. Proline and ATP binding cause respectively conformational changes in the proline binding loop and motif 2 loop. However formation of the activated intermediate is necessary for the final conformational ordering of a ten residue peptide ("ordering loop") close to the active site which would appear to be essential for functional tRNA 3' end binding. These induced fit conformational changes ensure that the enzyme is highly specific for proline activation and aminoacylation. We also present new structures of apo and AMP bound histidyl-tRNA synthetase (HisRS) from T. thermophilus which we compare to our previous structures of the histidine and histidyl-adenylate bound enzyme. Qualitatively, similar results to those observed with T. thermophilus prolyl-tRNA synthetase are found. However histidine binding is sufficient to induce the co-operative ordering of the topologically equivalent histidine binding loop and ordering loop. These two examples contrast with most other class II aminoacyl-tRNA synthetases whose pocket for the cognate amino acid side-chain is largely preformed. T. thermophilus prolyl-tRNA synthetase appears to be the second class II aminoacyl-tRNA synthetase, after HisRS, to use a positively charged amino acid instead of a divalent cation to catalyse the amino acid activation reaction.

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.

Refined crystal structure of the seryl-tRNA synthetase from Thermus thermophilus at 2.5 A resolution.

The three-dimensional structure of the seryl-tRNA synthetase from Thermus thermophilus has been determined and refined at 2.5 A resolution. The final model consists of a dimer of 421 residues each and 190 water molecules. The R-factor is 18.4% for all the data between 10 and 2.5 A resolution. The structure is very similar to that of the homologous enzyme from Escherichia coli, with an r.m.s. difference of 1.5 A for the 357 alpha-carbon atoms considered equivalent. The comparison of the two structures indicates increased hydrophobicity, reduced conformational entropy and reduced torsional strain as possible mechanisms by which thermostability is obtained in the enzyme from the thermophile.

Crystallization of the seryl-tRNA synthetase-tRNA(Ser) complex from Thermus thermophilus.

The complex between seryl-tRNA synthetase and its cognate tRNA from the extreme thermophile Thermus thermophilus has been crystallized from ammonium sulphate solutions. Two different tetragonal crystal forms have been characterized, both diffracting to about 6 A using synchrotron radiation. One form grows as large bipyramids and has cell dimensions a = b = 127 A, c = 467 A, and the second form occurs as long, thin square prisms with cell dimensions a = b = 101 A, c = 471 A. Analysis of washed and dissolved crystals demonstrates the presence of both protein and tRNA.

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

Crystals have been obtained of threonyl-tRNA synthetase from the extreme thermophile Thermus thermophilus using sodium formate as a precipitant. The crystals are very stable and diffract to at least 2.4 A. The crystals belong to space group P2(1)2(1)2(1) with cell parameters a = 61.4 A, b = 156.1 A, c = 177.3 A.

Immuno-chemical non-cross-reactivity between eukaryotic and prokaryotic seryl-tRNA synthetases.

Monospecific polyclonal antibodies (pAbs) against highly purified bovine seryl-tRNA synthetase (SerRS, EC 6.1.1.1) were prepared and their specificity tested. The interactions of pAbs with SerRS from different organisms were investigated by protein immunoblotting and ELISA methods. pAbs inhibit eukaryotic SerRS aminoacylating activity and exert no effect on SerRS activity from prokaryotes. It is proposed that prokaryotic and eukaryotic SerRS evolve from different ancestor genes.

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.

[Primary and spatial structure of tRNA]. / Pervichnaia i prostranstvennaia struktura tRNK.

The recent achievements in studying of structure of tRNA are considered in the present paper. A brief analysis of the new methods for sequencing tRNA was carried out. Due to the development of these methods about 300 tRNA primary structures have been determined. Comparison of the primary tRNA structures gives us the possibility to divide them into seven classes: prokaryotic initiator tRNAs and eukaryotic initiator tRNAs; prokaryotic elongator tRNAs and eukaryotic elongator tRNAs; archaebacterial tRNAs; and mitochondrial tRNAs of lower and higher eukaryotes. Structural properties of the tRNAs of each of these classes are discussed. The second part of the paper is devoted to the three-dimensional structure of tRNA. Recent data in this field obtained by X-ray crystallographic technique as well as by high-resolution NMR and chemical modification methods are reviewed.

[Primary structure of tRNALeuIAG from the mammary gland of the lactating cow]. / Pervichnaia struktura tRNKLeuIAG laktiruiushchei molochnoi zhelezy korov.

The nucleotide sequence of the tRNALeuIAG from a lactating cow mammary gland was determined by ultramicrospectrophotometrical method and rapid gel sequencing procedure. The chain length of this tRNA is 85 nucleotides, 15 of them including 6 psi, are modified nucleotides. The primary structure of tRNALeuIAG is absolutely identical to major species of leucyl tRNAIAG from bovine liver and differs in 21 positions from cow mammary gland tRNALeuCAG.

[A study of the conformation of tRNA(IAGLeu) from the cow mammary gland using chemical modification methods]. / Issledovanie élementov prostranstvennoi struktury tRNK(IAGLeu) molochnoi zhelezy korov metodami khimicheskoi modifikatsii.

The nucleosides of tRNA(IAGLeu) (with a long variable loop) from the cow mammary gland included in formation of the three-dimensional structure have been analysed by the chemical modification methods. Exposed guanosine and cytidine residues were detected by means of dimethylsulfate, whereas diethylpyrocarbonate was used to detect exposed adenosine residues. The low level of the modification was characteristic of guanosine residues in positions 10 (m2G), 13, 15, 23, 24, 29, 30, 47 H, 51, 52, 53, 57; of cytidine residues in positions 48 (m5C), 56 and those involved in Watson--Crick pairing; of adenosine residues in positions 14, 22, 31, 42, 59, 64. Most bases of tRNA(IAGLeu) thus detected are similarly located in the yeast tRNA(Phe) molecule, which suggests a common role of these bases in the formation of the spacial structure of both tRNAs.

[Determination of phosphate residues participating in the formation of the spatial structure of tRNA- Leu IAG from cow mammary glands]. / Opredelenie ostatkov fosfornoi kisloty, uchastvuiushchikh v obrazovanii prostranstvennoi struktury tRNK- Leu IAG molochnoi zhele korov.

The phosphates of the tRNA-Leu IAG from cow mammary gland (tRNA which has a long variable loop) participating in the formation of three-dimensional structure were studied by alkylation with ethylnitrosourea and methylnitrosourea. A low degree of modification was observed for the phosphates of the following nucleotides: 7, 8, 9, 10 (at the bend site between the acceptor and D-stem); 18, 19, 20A and 21 in the D-loop; 47H and 49 at the joint of variable and T-stem; 57, 58 and 59 in the T-loop.

[Primary structure of tRNA2Leu from the bovine udder. Determination of the oligonucleotide structure of the pyrimidyl-RNAse hydrolysate of tRNA2Leu by microspectrophotometric methods]. / Pervichnaia struktura tRNK2Leu molochnoi zhelezy korov. Opredelenie struktury oligonukleotidov pirimidil-RNKaznogo gidrolizata tRNK2Leu mikrospektrofotometricheskimi metodami.

tRNA2Leu from cow mammary gland has been degraded with pancreatic ribonuclease, and the fragments obtained were separated by DEAE-cellulose micro-column chromatography in 7 M urea at pH 7,5. Rechromatography was performed on a DEAE-cellulose micro-column at pH 3,7 and also on Dowex 1 X 2 in a formiate system. Nucleotide analysis was carried out with the aid of T2-RNase hydrolysis followed by chromatography on anion-exchanger AG 1 X 8. Nucleosides were separated on Aminex A-6 at pH 9,8. 15 minor components were shown to be present: T, 2 psi, 2Um, 2D, m5C, ac4C, m1G, 2m2G, m22G, m1A and N, the N is not identified so far. The structure of oligonucleotides was established by terminal analysis, hydrolysis with T1-RNase and also using incomplete hydrolysis by the snake venom phosphodiesterase.

[Primary structure of tRNA2Leu of bovine mammary gland. Oligonucleotides of T1-RNAse hydrolysate. Reconstruction of a total nucleotide sequence]. / Pervichnaia struktura tRNK2Leu molochnoi zhelezy korov. Oligonukleotidy T1-RNKaznogo gidrolizata. Rekonstruktsiia polnoi nukleotidnoi posledovatel'nosti.

The oligonucleotides obtained by digestion of tRNA2Leu from cow mammary gland with T1 RNase were separated by micro-column chromatography on DEAE-cellulose in 7 M urea at pH 7,5 and 3,7, and in addition on Dowex 1 x 2. The digest consisted of 18 individual components, the larger being a tridecanucleotide. Micro-column chromatography of nucleotides on anion-exchanger AG 1 x 8 and nucleosides on Aminex A-6 was used to determine the base composition of the oligonucleotides. The oligonucleotide structure was established using terminal analysis, hydrolysis by pancreatic and U2-RNases and incomplete hydrolysis by snake venom phosphodiesterases. The total primary structure of tRNA2Leu was derived from overlapping fragments isolated after its complete hydrolysis with pancreatic and T1 RNase and using data obtained on S1-nuclease digestion of tRNA. The methods of rapid gel-sequencing were also employed for checking the nucleotide sequence of tRNA2Leu from cow mammary gland.

[Comparative analysis of interaction sites of Thermus thermophilus and Escherichia coli tRNA(Tyr) with homologous aminoacyl-tRNA synthetases by means of chemical modification and nuclease hydrolysis]. / Sravnitel'nyi analiz uchastkov vzaimodeistviia tRNK(Tyr) iz Thermus thermophilus i Escherichia coli s gomologichnymi aminoatsil-tRNK-sintetazami metodami khimicheskoi modifikatsii i nukleaznogo gidroliza.

A nucleotide sequence of tRNA(Tyr) from the extreme thermophile Thermus thermophilus HB-27 living at 75 degrees C was determined. It is 86 nt long and shares a 52% homology with tRNA(Tyr) from Escherichia coli. A comparative analysis of the interaction sites of tRNA(Tyr) from T. thermophilus and E. coli with the cognate aminoacyl-tRNA synthetases was accomplished by the chemical modification and nuclease hydrolysis approaches. The tRNA(Tyr) was shown to interact with the cognate enzyme in the anticodon stem (on the 5'-side), in the anticodon, in the variable stem and loop (on the 5'-side), and in the acceptor stem (on the 3'-side). These regions are located in the variable stem of the L-form. It was demonstrated that, upon forming the complex E. coli tRNA(Tyr)-cognate synthetase, endonuclease V1 induces additional cleavages of phosphodiester bonds on the 3'-side of the anticodon stem and on the 5'-side of the T-stem. This implies that tRNA may change its conformation when it interacts with the enzyme.