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PubMed2000

tRNA aminoacylation by arginyl-tRNA synthetase: induced conformations during substrates binding.

The 2.2 A crystal structure of a ternary complex formed by yeast arginyl-tRNA synthetase and its cognate tRNA(Arg) in the presence of the L-arginine substrate highlights new atomic features used for specific substrate recognition. This first example of an active complex formed by a class Ia aminoacyl-tRNA synthetase and its natural cognate tRNA illustrates additional strategies used for specific tRNA selection. The enzyme specifically recognizes the D-loop and the anticodon of the tRNA, and the mutually induced fit produces a conformation of the anticodon loop never seen before. Moreover, the anticodon binding triggers conformational changes in the catalytic center of the protein. The comparison with the 2.9 A structure of a binary complex formed by yeast arginyl-tRNA synthetase and tRNA(Arg) reveals that L-arginine binding controls the correct positioning of the CCA end of the tRNA(Arg). Important structural changes induced by substrate binding are observed in the enzyme. Several key residues of the active site play multiple roles in the catalytic pathway and thus highlight the structural dynamics of the aminoacylation reaction.

Anticodon↗
PubMed1994

High level expression in E. coli and purification of yeast transcription factor IIIA.

Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form. High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E. coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species. Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.

Base Sequence↗
PubMed1988

Ordering promoter binding of class III transcription factors TFIIIC1 and TFIIIC2.

The separation of the mammalian class III transcription factor TFIIIC into two functional components, termed TFIIIC1 and TFIIIC2, enabled an analysis of their functions in transcription initiation. Template competition assays were used to define the order with which these factors interact in vitro to form stable preinitiation complexes on the adenovirus VAI and Drosophila melanogaster tRNA(Arg) genes. The interaction between these genes and TFIIIC2, the factor that binds with high affinity to the B block, was both necessary and sufficient for template commitment. When either the VAI or tRNA(Arg) gene was preincubated with TFIIIC2 alone, transcription of a second gene added subsequently was excluded, indicating that TFIIIC2 bound stably to the first template. Furthermore, the interaction between TFIIIC2 and these genes must occur prior to that of TFIIIC1 or TFIIIB. Once TFIIIC2 was bound, TFIIIC1 could bind to the tRNA(Arg) and VAI genes, although its interaction with the VAI gene was less stable than that with the tRNA(Arg) gene. TFIIIB activity bound stably to the complex of both genes with TFIIIC2. These results demonstrate that TFIIIC2 is the first transcription factor to bind to these genes and that TFIIIB and TFIIIC1 can then interact in either order to form a preinitiation complex.

Adenoviridae↗
PubMed1988

Translation of the sequence AGG-AGG yields 50% ribosomal frameshift.

We have inserted the sequence 5'-AAG-GAGGU-3', which is complementary to the 3' terminus of Escherichia coli 16S rRNA, in a reading frame and analyzed its effect on the accuracy and overall rate of translation in vivo. Translation over the sequence yields a 50% ribosomal frameshift if the reading phase is A-AGG-AGG-U. The other two possible frames do not give shifts. The introduction of a UAA stop codon before (UAA-AGG-AGG-U) but not after (A-AGG-AGG-UAA) the AGG codons abolishes the frameshift. The change in the reading phase occurs exclusively to the +1 direction. Efficient frameshifting is also induced by the sequence A-AGA-AGA-U. The arginine codons AGG and AGA are read by minor tRNA. Suppression of frameshifting takes place when a gene for minor tRNA(Arg) is introduced on a multicopy plasmid. We suggest that frameshifting during translation of the A-AGG-AGG-U sequence is due to the erroneous decoding of the tandem AGG codons and arises by depletion of tRNA(Arg). The complementarity of tandem AGG codons to the 3' terminus of 16S rRNA is a coincidence and apparently not related to the shift. Replacing the AGG-AGG sequence by the optimal arginine codons CGU-CGU does not increase the overall rate of translation.

Base Sequence↗
PubMed1991

Cloning and expression in vitro of a gene encoding tRNAArgACG from the nematode Caenorhabditis elegans.

A gene (rtr-1) coding for the tRNAArgACG has been isolated and characterized from the nematode, Caenorhabditis elegans. The coding portion is not interrupted by an intron and is followed by a track of four thymidines associated with termination by RNA polymerase III. The predicted mature product is 76 nucleotides (nt) long including the CCA tail, and is specific for the most used Arg codon in C. elegans. The gene can be transcribed and processed in a homologous in vitro system. The 82-nt primary transcript begins at the first purine upstream from the mature tRNA 5' end and terminates after the first thymidine of the terminator signal.

Animals↗
PubMed2006

The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability.

Although the La protein stabilizes nascent pre-tRNAs from nucleases, influences the pathway of pre-tRNA maturation, and assists correct folding of certain pre-tRNAs, it is dispensable for growth in both budding and fission yeast. Here we show that the Saccharomyces cerevisiae La shares functional redundancy with both tRNA modification enzymes and other proteins that contact tRNAs during their biogenesis. La is important for growth in the presence of mutations in either the arginyl tRNA synthetase or the tRNA modification enzyme Trm1p. In addition, two pseudouridine synthases, PUS3 and PUS4, are important for growth in strains carrying a mutation in tRNA(Arg)(CCG) and are essential when La is deleted in these strains. Depletion of Pus3p results in accumulation of the aminoacylated mutant tRNA(Arg)(CCG) in nuclei, while depletion of Pus4p results in decreased stability of the mutant tRNA. Interestingly, the degradation of mutant unstable forms of tRNA(Arg)(CCG) does not require the Trf4p poly(A) polymerase, suggesting that yeast cells possess multiple pathways for tRNA decay. These data demonstrate that La functions redundantly with both tRNA modifications and proteins that associate with tRNAs to achieve tRNA structural stability and efficient biogenesis.

Amino Acyl-tRNA Synthetases↗
PubMed1988

Cloning and sequence analysis of an XbaI fragment of rainbow trout mitochondrial DNA.

A 2.4 kbp XbaI fragment of rainbow trout mitochondrial DNA was cloned into pTZ18R. DNA sequence analysis reveals that this segment of the genome encodes URF3, tRNA(Arg), URF4L and URF4 in the same orientation as other vertebrate mitochondrial genomes. Comparison of these segments of the rainbow trout mitochondrial genome with the corresponding sequences in human mitochondrial DNA shows that approximately 60% of the nucleotides are the same in both species.

Amino Acid Sequence↗
PubMed1998

Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon.

The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobus sp. strain 7, a thermoacidophilic archaeon, was cloned and sequenced. An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues. The deduced sequence was supported by partial amino acid sequence analyses. All the catalytically important residues were conserved. A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function. Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E. coli. Coexpression of tRNA(Arg), cognate for the rare codon AGA in E. coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obtained after removal of heat denatured E. coli proteins.

Amino Acid Sequence↗
PubMed1998

[Mutation induced by space conditions in Escherichia coli strains].

To study the mutagenesis of microorganism under space conditions, three Escherichia coli strains CSH108, A2 and A3, were flown on board the Chinese recoverable satellite (JB1 - B9611020) in Oct . 1996. Three ways of sample boarding were designed . After recovery, survival and frequency of lacI- mutation and Arg+ reversion were determined in the flight strains. The results showed that: E. coli strains could survive after the flight conditions. The lacI- mutation frequency of A3 strain in the biocabin was sixty - seven times that of the ground control, the Arg+ reversion frequency of CSH108 in the lead chamber, was ten times that of the ground control; and among the revertants the mutation frequency of non-sense suppressor increased remarkably. The results indicate that the space conditions may greatly enhance the mutation frequency of certain genes in the microorganism and may provide an effective method for microorganism breeding.

Arginine↗
PubMed2001

A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice.

The pathophysiologic pathways and clinical expression of mitochondrial DNA (mtDNA) mutations are not well understood. This is mainly the result of the heteroplasmic nature of most pathogenic mtDNA mutations and of the absence of clinically relevant animal models with mtDNA mutations. mtDNA mutations predisposing to hearing impairment in humans are generally homoplasmic, yet some individuals with these mutations have severe hearing loss, whereas their maternal relatives with the identical mtDNA mutation have normal hearing. Epidemiologic, biochemical and genetic data indicate that nuclear genes are often the main determinants of these differences in phenotype. To identify a mouse model for maternally inherited hearing loss, we screened reciprocal backcrosses of three inbred mouse strains, A/J, NOD/LtJ and SKH2/J, with age-related hearing loss (AHL). In the (A/J x CAST/Ei) x A/J backcross, mtDNA derived from the A/J strain exerted a significant detrimental effect on hearing when compared with mtDNA from the CAST/Ei strain. This effect was not seen in the (NOD/LtJ x CAST/Ei) x NOD/LtJ and (SKH2/J x CAST/Ei) x SKH2/J backcrosses. Genotyping revealed that this effect was seen only in mice homozygous for the A/J allele at the Ahl locus on mouse chromosome 10. Sequencing of the mitochondrial genome in the three inbred strains revealed a single nucleotide insertion in the tRNA-Arg gene (mt-Tr) as the probable mediator of the mitochondrial effect. This is the first mouse model with a naturally occurring mtDNA mutation affecting a clinical phenotype, and it provides an experimental model to dissect the pathophysiologic processes connecting mtDNA mutations to hearing loss.

Age Factors↗
PubMed1994

Enhancement of expression of human granulocyte-macrophage colony stimulating factor by argU gene product in Escherichia coli.

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned into expression vector pIN III-ompA1 and expressed in Escherichia coli JA221. When supplementation with a minor tRNA(AGA/AGG)Arg encoded by the E. coli argU gene, the expression level of hGM-CSF was raised about 3-4-fold, although there is only one rare AGG codon in hGM-CSF cDNA gene.

Base Sequence↗
PubMed1992

A reactive nucleophile proximal to vicinal thiols is an evolutionarily conserved feature in the mechanism of Arg aminoacyl-tRNA protein transferase.

Aminoacyl-tRNA protein transferases post-translationally aminoacylate protein N-termini. At least in part, these enzymes function to allow a subset of cellular proteins to be targeted for protein degradation. A eukaryotic enzyme of this class, Arg aminoacyl-tRNA protein transferase, arginylates N-terminal Glu or Asp residues of proteins, allowing such proteins to be recognized by a specific ubiquitin-protein ligase. We showed previously that inorganic arsenite, a reagent expected to bind specifically to protein vicinal thiol groups, inhibited Arg aminoacyl-tRNA transferase activity in rabbit reticulocyte lysate (N. S. Klemperer and C. M. Pickart, 1989, J. Biol. Chem. 264, 19245-19252). We now report that a bifunctional arsenoxide reagent, p-[(bromoacetyl)-amino]phenylarsenoxide, is a potent and irreversible inactivator of the same enzyme (K0.5 = 11.5 microM). Bromoacetyl aniline, which lacks the arsenoxide moiety, has no effect. These results show that the transferase has a reactive nucleophile proximal to the site which binds arsenoxides. The related monofunctional arsenoxide reagent, p-aminophenylarsenoxide, is a reversible inhibitor whose potency (K0.5 = 7.7 microM) is 20-fold greater than that of inorganic arsenite. As expected for a mechanism in which p-aminophenylarsenoxide binds to vicinal thiol groups: (i) pretreatment of reticulocyte lysate with a thiol-blocking reagent prevents binding of the transferase to a phenylarsenoxide-Sepharose column; and (ii) inhibition by p-aminophenylarsenoxide is reversed by a competing chemical dithiol, but not by a monothiol reagent. Like the rabbit enzyme, Arg aminoacyl-tRNA protein transferase from the yeast Saccharomyces cerevisiae (expressed in Escherichia coli) is reversibly inhibited by the monofunctional phenylarsenoxide and irreversibly inactivated by the bifunctional phenylarsenoxide (but not by bromoacetylaniline). Thus, a reactive nucleophile proximal to vicinal thiol groups is a conserved feature of the activity of the transferase. We speculate that these groups are catalytic elements in the transferase active site.

Acyltransferases↗
PubMed2000

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops.

Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAs(Arg) including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2',3'-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAs(Arg), the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNA(Arg)-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAs(Arg), which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.

Anticodon↗
PubMed2000

Cotranscription of 5S rRNA-tRNA(Arg)(ACG) from Brassica napus chloroplasts and processing of their intergenic spacer.

S1 mapping showed that at least a significant portion of the 5S rRNA and tRNA(Arg)(ACG) is co-transcribed in canola chloroplast, making trnR the last gene transcribed in an operon of which the final sequence is 5'-16S-tRNA(Ile)-tRNA(Ala)-23S-4.5S-5S-tRNA(Arg)-3'. Various RNA termini representing RNA processing sites at several parts of the 5S rRNA-tRNA(Arg) area were detected. This gene spacer is substantially conserved among various species compared here, and a secondary structure model for this chloroplast region in canola applies to other plant sequences. The conservation of this intergenic sequence suggests a functional role, possibly by providing recognition structures for endogenous RNases involved in its maturing process.

Base Sequence↗
PubMed1995

Synthesis of cysteine-containing dipeptides by aminoacyl-tRNA synthetases.

Arginyl-tRNA synthetase (ArgRS) catalyses AMP- and PPi-independent deacylation of Arg-tRNAArg in the presence of cysteine. A dipeptide, Arg-Cys, is a product of this deacylation reaction. Similar reaction with homocysteine yields Arg-Hcy. Arginine is a noncompetitive inhibitor of the cysteine-dependent deacylation which indicates that cysteine binds to the enzyme-Arg-tRNAArg complex at a site separate from the arginine binding site. In the presence of arginine, [14C]Arg-tRNAArg is deacylated at a rate similar to the rate of its spontaneous deacylation in solution and [14C]arginine is a product. Experiments with cysteine derivatives indicate that the -SH group is essential for the reaction whereas -NH2 and -COOH groups are not. Thioesters of arginine are formed with 3-mercaptopropionic acid, N-acetyl-L-cysteine and dithiothreitol. These data suggest that formation of the dipeptide Arg-Cys involves a thioester intermediate, S-(L-arginyl)-L-cysteine, which is not observed because of the rapid rearrangement to form a stable peptide bond. Facile intramolecular reaction results from the favorable geometric arrangement of the alpha-amino group of cysteine with respect to the thioester formed in the initial reaction. Similar reactions, yielding Ile-Cys and Val-Cys, are catalyzed by isoleucyl- and valyl-tRNA synthetases, respectively.

Amino Acyl-tRNA Synthetases↗
PubMed2003

Substrate-induced conformational changes in Escherichia coli arginyl-tRNA synthetase observed by 19F NMR spectroscopy.

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.

Anticodon↗
PubMed2003

Arginyl-tRNA synthetase with signature sequence KMSK from Bacillus stearothermophilus.

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.

Adenosine Triphosphatases↗