ABSTRACT
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.
Subject(s)
Archaea/enzymology , Archaea/genetics , Leucine-tRNA Ligase/chemistry , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , RNA, Transfer/chemistry , Hydrolysis , Leucine-tRNA Ligase/metabolism , Nucleic Acid Conformation , Protein Binding , RNA Editing , RNA, Transfer/genetics , Substrate SpecificityABSTRACT
Maintenance of amino acid specificity by aminoacyl-tRNA synthetases, particularly prolyl-tRNA synthetase, requires for not only specific recognition of homologic amino acid, but also missynthesized products hydrolysis, known as editing. The speeding-up of the enzymatic hydrolysis of missynthesized alanyl adenylate by bacteria Enterococcus faecalis prolyl-tRNA synthetase in the presence of tRNAPro, and also importance for this function of 2'- and 3'-hydroxyle groups of tRNA 3'-terminal adenosine ribose is shown in the work. Furthermore, results are shown, that support the absence of editing (INS) domain role in alanyl adenylate hydrolysis. Possible significance of tRNA-dependent alanyl adenylate hydrolysis by prolyl-tRNA synthetase for prolyl-tRNAPro synthesis specificity maintenance is discussed.
Subject(s)
Amino Acyl-tRNA Synthetases/antagonists & inhibitors , Amino Acyl-tRNA Synthetases/genetics , Enterococcus faecalis/enzymology , RNA Editing , RNA, Transfer/genetics , Transfer RNA Aminoacylation , Adenosine Triphosphate/metabolism , Enterococcus faecalis/genetics , Hydrolysis , Mutagenesis, Site-Directed , Protein Structure, Secondary , Rhodopseudomonas/enzymology , Rhodopseudomonas/geneticsABSTRACT
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.
Subject(s)
Phosphoric Acids/chemistry , RNA, Transfer, Leu/chemistry , RNA, Transfer, Ser/chemistry , Thermus thermophilus/genetics , Anticodon , Base Sequence , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Transfer, Leu/genetics , RNA, Transfer, Ser/geneticsABSTRACT
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.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Escherichia coli/chemistry , RNA, Bacterial/chemistry , RNA, Transfer, Tyr/chemistry , Ribonucleases/chemistry , Thermus thermophilus/chemistry , Autoradiography , Base Sequence , Escherichia coli/genetics , Hydrolysis , Molecular Sequence Data , Sequence Homology, Nucleic Acid , Thermus thermophilus/geneticsABSTRACT
Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNAlys or Escherichia coli tRNAlys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18-22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 A, c = 119 A) and diffract to at least 2.7 A resolution. However the homocomplex is of space group P42(1)2 and the heterocomplex of space group I422.
Subject(s)
Escherichia coli/genetics , Lysine-tRNA Ligase/chemistry , RNA, Transfer, Lys/chemistry , Thermus thermophilus/enzymology , Thermus thermophilus/genetics , Crystallization , Crystallography, X-Ray , RNA, Bacterial/chemistry , Temperature , Time FactorsABSTRACT
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.
Subject(s)
Serine-tRNA Ligase/ultrastructure , Thermus thermophilus/enzymology , Amino Acid Sequence , Bacterial Proteins/ultrastructure , Crystallography, X-Ray , Escherichia coli/enzymology , Fungal Proteins/ultrastructure , Hot Temperature , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Protein Denaturation , Protein Structure, Tertiary , Saccharomyces cerevisiae/enzymology , Sequence Alignment , Sequence Homology , Surface PropertiesABSTRACT
Interaction of the bovine liver tRNA(GCUSer) having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by alkylation with ethylnitrosourea. It was shown that seryl-tRNA synthetase protects 3'-phosphates of nucleotides 12, 13 in D-stem and 45-47-, 47 G.-, 47 H-variable stem of tRNA(GCUreS) from alkylation. An anticodon loop of tRNA(GCUSer) did not interact with seryl-tRNA synthetase.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Liver/metabolism , RNA, Transfer, Ser/metabolism , Alkylation , Amino Acyl-tRNA Synthetases/drug effects , Animals , Binding Sites , Cattle , Drug Interactions , Ethylnitrosourea/pharmacology , In Vitro Techniques , Liver/drug effects , RNA, Transfer, Ser/drug effects , RNA, Transfer, Ser/isolation & purificationABSTRACT
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.
Subject(s)
RNA, Transfer, Ser/chemistry , Serine-tRNA Ligase/chemistry , Thermus thermophilus/chemistry , Crystallization , Electrophoresis, Polyacrylamide Gel , RNA, Transfer, Ser/metabolism , Serine-tRNA Ligase/metabolism , Thermus thermophilus/enzymology , X-Ray DiffractionABSTRACT
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.
Subject(s)
RNA, Transfer, Ser/chemistry , Serine-tRNA Ligase/chemistry , Thermus thermophilus/enzymology , Crystallization , Electrophoresis, Polyacrylamide Gel , RNA, Transfer, Ser/metabolism , Serine-tRNA Ligase/metabolism , X-Ray DiffractionABSTRACT
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.
Subject(s)
Serine-tRNA Ligase/metabolism , Animals , Antibodies , Blotting, Western , Cattle , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Escherichia coli/enzymology , Immunohistochemistry , Liver/enzymology , Rabbits , Serine-tRNA Ligase/immunologyABSTRACT
The interaction of the cow mammary gland tRNA(IAGLeu), having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by the alkylation with ethylnitrosourea. It was shown that leucyl-tRNA synthetase protects from alkylation 3'-phosphates of the nucleotides 12-13 in D-loop, 23-24 in D-stem and 37-43 in the anticodon arm of tRNA(IAGLeu). All regions of interaction with the aminoacyl-tRNA synthetase are located in the same plane of tRNA whereas the long variable loop is in another plane.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Mammary Glands, Animal/metabolism , RNA, Transfer, Leu/genetics , Alkylation , Animals , Autoradiography , Base Sequence , Cattle , Electrophoresis, Polyacrylamide Gel , Female , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Transfer, Leu/metabolismABSTRACT
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.
Subject(s)
Amino Acyl-tRNA Synthetases/isolation & purification , Thermus/enzymology , Threonine-tRNA Ligase/isolation & purification , Crystallization , Enzyme Stability , Protein Conformation , X-Ray DiffractionABSTRACT
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.
Subject(s)
Amino Acyl-tRNA Synthetases , Serine-tRNA Ligase , Thermus/enzymology , X-Ray DiffractionABSTRACT
A method for isolating tyrosyl-tRNA synthetase from Thermus thermophilus is described, including ammonium sulfate fractionation, chromatography on DEAE-sepharose, hydroxyapatite, heparin-sepharose and hydrophobic chromatography on Toyopearl HW-65. The yield of the purified enzyme was 1.6 mg per 1 kg of T. thermophilus cells. The enzyme is a dimer protein of the alpha 2 type with molecular weight of 100 kDa.
Subject(s)
Amino Acyl-tRNA Synthetases/isolation & purification , Thermus/enzymology , Tyrosine-tRNA Ligase/isolation & purification , Catalysis , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Molecular Weight , TemperatureABSTRACT
The primary structure of tRNA(1Ser) from the bovine liver has been studied. pG- A-C-G-A-G-G-U-G-G-C-ac4C-G-A-G-D-Gm-G-D-D-A-A-G-G- C-m2(2)-G-A-psi-G-G-A-m3C-U-G-C-U-A*-A-psi-C-C-A-U-Um-G-psi- G-C-U-m3C-U-G-C-A-C-G-m5C-G-U-G-G-G-T-psi-C-G-m1A-A- U-C-C-C-A-U-C-C-U-C-G-U-C-G-C-C-AOH. A comparison of the nucleotide sequence of tRNA(1Ser) from the bovine liver with already known sequences of serine tRNA revealed a number of common nucleotides, some of them, probably, participated in specific interaction with seryl-tRNA synthetase.
Subject(s)
Base Sequence , Liver/analysis , RNA, Transfer, Amino Acid-Specific/analysis , RNA, Transfer, Ser/analysis , Sequence Homology, Nucleic Acid , Animals , Cattle , Hydrolysis , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides/analysisABSTRACT
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.
Subject(s)
Mammary Glands, Animal/analysis , Nucleic Acid Conformation , RNA, Transfer, Amino Acid-Specific/analysis , RNA, Transfer, Leu/analysis , Animals , Base Composition , Cattle , Chemical Phenomena , Chemistry , Nucleic Acid DenaturationABSTRACT
A new approach allowing detection of contact points between RNAs and proteins has been developed using trans-diamminedichloroplatinum(II) as the cross-linking reagent. The advantage of the method relies on the fact that the coordination bonds between platinum and the potential acceptors on proteins and nucleic acids (mainly S of cysteine or methionine residues; N of imidazole rings in histidine residues; N7 of guanine, N1 of adenine, and N3 of cytosine residues) can be reversed, so that the cross-linked oligonucleotides or peptides in contact within a complex can be analyzed directly. The method was worked out with the ribosome from Escherichia coli and the tRNAVal/valyl-tRNA synthetase system from the yeast Saccharomyces cerevisiae. In the first system the platinum approach permitted detection of ribosomal proteins cross-linked to 16S rRNA within the 30S subunits (mainly S18 and to a lower extent S3, S4, S11, and S13/S14); in the second system major oligonucleotides of tRNAVal cross-linked to valyl-tRNA synthetase were detected in the anticodon stem and loop, in the variable loop, and in the 3' terminal amino acid accepting region. These results are discussed in light of the current knowledge on ribosome and tRNAs and of potential applications of the methodology.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Cisplatin/pharmacology , Cross-Linking Reagents , RNA, Transfer, Amino Acyl/metabolism , Valine-tRNA Ligase/metabolism , Base Sequence , Escherichia coli/metabolism , Kinetics , Nucleic Acid Conformation , Ribosomes/metabolismABSTRACT
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.
