ABSTRACT
Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.
Subject(s)
Arginine-tRNA Ligase/chemistry , Cytosol/enzymology , Leucine-tRNA Ligase/chemistry , Adenosine Triphosphate/chemistry , Amino Acid Sequence , Animals , Arginine-tRNA Ligase/metabolism , Baculoviridae/genetics , Cell Line , Cloning, Molecular , Cytosol/metabolism , DNA Primers/chemistry , DNA, Complementary/metabolism , Green Fluorescent Proteins/metabolism , Humans , Insecta , Kinetics , Leucine/chemistry , Leucine-tRNA Ligase/metabolism , Macromolecular Substances , Microscopy, Confocal , Molecular Sequence Data , Phosphates/chemistry , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , RNA, Transfer/chemistry , Sequence Homology, Amino Acid , Subcellular Fractions , TemperatureABSTRACT
The human mitochondrial tRNALeu(CUN) [hmtRNALeu(CUN)] corresponds to the most abundant codon for leucine in human mitochondrial protein genes. Here, in vitro studies reveal that the U48C substitution in hmtRNALeu(CUN), which corresponds to the pathological T12311C gene mutation, improved the aminoacylation efficiency of hmtRNALeu(CUN). Enzymatic probing suggested a more flexible secondary structure in the wild-type hmtRNALeu(CUN) transcript compared with the U48C mutant. Structural analysis revealed that the flexibility of hmtRNALeu(CUN) facilitates a T-stem slip resulting in two potential tertiary structures. Several rationally designed tRNALeu(CUN) mutants were generated to examine the structural and functional consequences of the T-stem slip. Examination of these hmtRNALeu(CUN) mutants indicated that the T-stem slip governs tRNA accepting activity. These results suggest a novel, self-regulation mechanism of tRNA structure and function.
Subject(s)
RNA, Transfer, Leu/chemistry , RNA, Transfer, Leu/metabolism , RNA/chemistry , RNA/metabolism , Transfer RNA Aminoacylation , Base Sequence , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Point Mutation , RNA/genetics , RNA, Mitochondrial , RNA, Transfer, Leu/geneticsABSTRACT
The mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS) is a rare congenital disorder of mitochondrial DNA. Five single nucleotide substitutions within the human mitochondrial tRNALeu(UUR) gene have been reported to be associated with MELAS. Here, we provide in vitro evidence that the aminoacylation capacities of these five hmtRNALeu(UUR) transcripts are reduced to different extents relative to the wild-type hmtRNALeu(UUR) transcript. A thermal denaturation experiment showed that the A3243G and T3291C mutants, which were the least charged by LeuRS, have fragile structures. In addition, the T3291C mutant can inhibit aminoacylation of the wild-type hmtRNALeu(UUR), indicating that it may act as an inhibitor in the mitochondrial heteroplasmic environment.
Subject(s)
DNA, Mitochondrial , MELAS Syndrome/genetics , Mitochondria/genetics , RNA, Transfer, Leu , Aminoacylation , Base Sequence , Hot Temperature , Humans , Mitochondria/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Oxidation-Reduction , Point Mutation , Protein Denaturation , RNA, Transfer, Leu/chemistry , RNA, Transfer, Leu/genetics , RNA, Transfer, Leu/metabolismABSTRACT
Previous investigations show that tRNA(Arg)-induced conformational changes of arginyl-tRNA synthetase (ArgRS) Omega-loop region (Escherichia coli (E. coli), Ala451-Ala457) may contribute to the productive conformation of the enzyme catalytic core, and E. coli tRNA(2)(Arg)(ICG)-bound and -free conformations of the Omega-loop exchange at an intermediate rate on NMR timescale. Herein, we report that E. coli ArgRS catalyzes tRNA(2)(Arg)(ICG) and tRNA(4)(Arg)(UCU) with similar efficiencies. However, 19F NMR spectroscopy of 4-fluorotryptophan-labeled E. coli ArgRS reveals that the tRNA(4)(Arg)(UCU)-bound and -free conformations of the Omega-loop region interconvert very slowly and the lifetime of bound conformation is much longer than 0.33 ms. Therefore, tRNA(4)(Arg)(UCU) differs from tRNA(2)(Arg)(ICG) in the conformation-exchanging rate of the Omega-loop. Comparative structure model of E. coli ArgRS is presented to rationalize these 19F NMR data. Our 19F NMR and catalytic assay results suggest that the tRNA(Arg)-induced conformational changes of Omega-loop little contribute to the productive conformation of ArgRS catalytic core.
Subject(s)
Arginine-tRNA Ligase/chemistry , Arginine-tRNA Ligase/metabolism , Escherichia coli/enzymology , RNA, Transfer, Arg/metabolism , Arginine/metabolism , Catalytic Domain , Kinetics , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation , RNA, Transfer, Arg/genetics , Tryptophan/analogs & derivatives , Tryptophan/metabolismABSTRACT
ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNA(Arg)s with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS ( argS ') was amplified by PCR and the gene encoding tRNA(Arg) (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNA(Arg) (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNA(Arg)s from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys(382) and Lys(385) of the KMSK sequence and at Gly(136) upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys(385) and Gly(136) for the amino acid-activation activity of B. stearothermophilus ArgRS.
Subject(s)
Arginine-tRNA Ligase/chemistry , Arginine-tRNA Ligase/metabolism , Geobacillus stearothermophilus/enzymology , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Arginine-tRNA Ligase/genetics , Circular Dichroism , Cloning, Molecular , Escherichia coli/metabolism , Glycine/genetics , Hot Temperature , Kinetics , Lysine/genetics , Molecular Sequence Data , Mutation , RNA, Transfer, Arg/genetics , RNA, Transfer, Arg/metabolismABSTRACT
The 19F nuclear magnetic resonance (NMR) spectra of 4-fluorotryptophan (4-F-Trp)-labeled Escherichia coli arginyl-tRNA synthetase (ArgRS) show that there are distinct conformational changes in the catalytic core and tRNA anticodon stem and loop-binding domain of the enzyme, when arginine and tRNA(Arg) are added to the unliganded enzyme. We have assigned five fluorine resonances of 4-F-Trp residues (162, 172, 228, 349 and 446) in the spectrum of the fluorinated enzyme by site-directed mutagenesis. The local conformational changes of E. coli ArgRS induced by its substrates observed herein by 19F NMR are similar to those of crystalline yeast homologous enzyme.
Subject(s)
Arginine-tRNA Ligase/chemistry , Arginine-tRNA Ligase/metabolism , Escherichia coli/enzymology , Tryptophan/analogs & derivatives , Anticodon/genetics , Arginine/metabolism , Binding Sites , Catalytic Domain , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Protein Conformation , Protein Structure, Secondary , RNA, Transfer, Arg/metabolism , Substrate SpecificityABSTRACT
The processing of human mitochondrial leucyl-tRNA synthetase had been previously investigated in insect cell. In the present work, the gene encoding human mitochondrial leucyl-tRNA synthetase with the same N-terminus as that processed in the mitochondria of insect cell was cloned and expressed in Escherichia coli. The enzyme was purified by affinity chromatography on Ni-NTA column. About 6 mg of human mitochondrial leucyl-tRNA synthetase was obtained from 1 liter of culture. The specific activity of the purified enzyme is 127.7 units/mg, the highest activity of the reported results; this enzyme has the potential for characterizing the mitochondrial tRNA mutants associated with some human mitochondrion-related neuromuscular disorders. The kinetic constants for three substrates: leucine, ATP, and E. coli tRNA1Leu (CAG) in the leucylation reaction are also reported herein.
Subject(s)
Escherichia coli , Leucine-tRNA Ligase/genetics , Leucine-tRNA Ligase/metabolism , Mitochondria/enzymology , Recombinant Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Genetic Engineering , Humans , Kinetics , Leucine-tRNA Ligase/biosynthesis , Leucine-tRNA Ligase/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWTKEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme purified from mitochondria catalyzed the leucylation of Escherichia coli tRNA(1)(Leu)(CAG) and Aquifex aeolicus tRNA(Leu)(GAG) with higher catalytic activity in the leucylation of E. coli tRNA(Leu) than that previously expressed in E. coli without the N-terminal 21 residues.
