Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.1.1.18 (glutaminyl-tRNA synthetase)
231 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have characterized the in vivo and in vitro transcription products of the Escherichia coli glnS gene which codes for the enzyme glutaminyl-tRNA synthetase. The in vivo glnS transcript is about 1.9 kilobases long. Sequence analysis of the 5'- and 3'-ends of glnS mRNA showed that transcription initiates approximately 30 bases upstream from the translation initiation codon AUG and terminates approximately 230 bases downstream from the termination codon UAA. Characterization of the in vitro transcripts of glnS revealed similar transcription initiation and termination sites as were found in the glnS mRNA produced in vivo. These results indicate that the Pribnow box structure upstream and the dyad symmetry terminator structure downstream of the glnS structural region are regulatory signals used for glnS expression. In vitro transcription of glnS is not autogenously regulated by glutaminyl-tRNA synthetase and glutamine; it is also not affected by the presence of tRNA.
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PMID:In vivo and in vitro transcription of the Escherichia coli glutaminyl-tRNA synthetase gene. 608 62

Saccharomyces cerevisiae glutaminyl-tRNA synthetase mutants were isolated through systematic screening of tight Gln- derivatives of a leaky glutamine auxotroph. These mutations define a single nuclear gene, GLN4. The gln4-1 mutation is specific for Gln-tRNA synthetase and shows a dosage effect in heterozygous diploids. The wild-type Gln-tRNA synthetase exhibits a Km for glutamine of 25 microM; the gln4-1 mutation increases this value 20-fold. These observations strongly suggest that GLN4 encodes the Gln-tRNA synthetase.
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PMID:Identification of a glutaminyl-tRNA synthetase mutation Saccharomyces cerevisiae. 614 64

We have isolated mutations in the Escherichia coli glnS gene encoding glutaminyl-tRNA synthetase [GlnS; L-glutamine:tRNAGln ligase (AMP-forming), EC 6.1.1.18] that give rise to gene products with altered specificity for tRNA and are designated "mischarging" enzymes. These were produced by nitrosoguanine mutagenesis of the glnS gene carried on a transducing phage (lambda pglnS+). We then selected for mischarging of su+3 tRNATyr with glutamine by requiring suppression of a glutamine-requiring beta-galactosidase amber mutation (lacZ1000). Three independently isolated mutants (glnS7, glnS8, and glnS9) were characterized by genetic and biochemical means. The enzymes encoded by glnS7, glnS8, and glnS9 appear to be highly selective for su+3 tRNATyr, because in vivo mischarging of other amber suppressor tRNAs was not detected. The GlnS mutants described here retain their capacity to correctly aminoacylate tRNAGln. All three independently isolated mutant genes encode proteins with isoelectric points that differ from those of the wild-type enzyme but are identical to each other. This suggests that only a single site in the enzyme structure is altered to give the observed mischarging properties. In vitro aminoacylation reactions with purified GlnS7 protein show that this enzyme can also mischarge some tRNA species lacking the amber anticodon. This is an example of mischarging phenotype conferred by a mutation in an aminoacyl-tRNA synthetase gene; the results are discussed in the context of earlier genetic studies with mutant tRNAs.
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PMID:Transfer RNA mischarging mediated by a mutant Escherichia coli glutaminyl-tRNA synthetase. 638 58

Escherichia coli glutaminyl-tRNA synthetase (GlnRS) (EC 6.1.1.18) is a monomeric polypeptide of 553 amino acids. Its amino acid sequence and its gene (glnS) sequence are known. A structural gene mutation, glnS7, codes for a mischarging GlnRS, which acylates some noncognate tRNA species (e.g., su+3 tRNATyr) with glutamine. The mutant enzyme was shown to catalyze in vitro the acylation of glutamine to su+3 tRNATyr, but not to wild-type tRNATyr. The mutation responsible produces an amino acid change in the amino-terminal half of the enzyme. Unexpectedly, overproduction of wild-type GlnRS also leads to in vivo mischarging of su+3 tRNATyr. In vitro and in vivo studies have not revealed evidence for an attenuation or autogenous regulation mechanism for GlnRS, but have implicated transcriptional and translational control in the expression of this enzyme.
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PMID:Misaminoacylation by glutaminyl-tRNA synthetase: relaxed specificity in wild-type and mutant enzymes. 638 80

Glutaminyl-tRNA synthetase has been purified by a simple, two-column procedure from an Escherichia coli K12 strain carrying the glnS structural gene on plasmid pBR322. The primary sequence of this enzyme as derived from the DNA sequence (see accompanying paper) has been confirmed. Manual Edman degradation was used to identify the NH2-terminal sequence of the protein. Oligopeptides scattered throughout the primary sequence of glutaminyl-tRNA synthetase were sequenced by the gas chromatographic-mass spectrometric method and matched to the theoretical peptides derived from the translated DNA sequence. The expected carboxyl terminus at position 550 was verified by carboxypeptidase B digestion. The primary sequence of glutaminyl-tRNA synthetase contains no extensive sequence repeats. A search was made for sequence homologies between this enzyme and the few other aminoacyl-tRNA synthetases for which primary sequences are available. A single homologous region is shared by at least three of the synthetases examined here.
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PMID:Escherichia coli glutaminyl-tRNA synthetase. II. Characterization of the glnS gene product. 674 44

Glutaminyl-tRNA synthetase from Escherichia coli has been purified to homogeneity with a yield of about 50%. It is a monomer of about 69 000 daltons. Arginyl and glutamyl-tRNA synthetases are also monomeric synthetases of molecular weight significantly lower than 100 000. In addition it is well known that these three synthetases require their cognate tRNA to catalyze the [32P]PPi-ATP exchange. Like arginyl-tRNA synthetase, but unlike glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase seems to contain some repeated sequences. Therefore no correlation can be established between the tRNA requirement of these synthetases for the catalysis of the isotope-exchange and the presence or the absence of sequence duplication. In the native enzyme four sulfhydryl groups react with dithiobisnitrobenzoic acid causing a loss of both the aminoacylation and the [32P]PPi-ATP exchange activities. The rate-limiting steps of the overall aminoacylation and its reverse reaction correspond, respectively, to the catalysis of the aminoacylation of tRNA Gln and of the the deacylation of glutaminyl-tRNA Gln. At acidic pH, glutaminyl-tRNA synthetase catalyzes the synthesis of the glutaminyl-tRNA Gln and its deacylation at significantly lower rates than the [32P]PPi-ATP exchange, indicating than glutaminyl-tRNA Gln cannot be an obligatory intermediate in this isotope exchange. These results suggest the existence of a two-step aminoacylation mechanism catalyzed by this enzyme.
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PMID:The glutaminyl-transfer RNA synthetase of Escherichia coli. Purification, structure and function relationship. 698 2

A variety of genetic, biochemical and structural studies have been used to determine factors ensuring the accuracy of recognition by aminoacyl-tRNA synthetases for tRNA. The identity elements of Escherichia coli tRNA(Gln) are located mainly in the anticodon and acceptor stem, and ensure the accurate recognition of the tRNA by glutaminyl-tRNA synthetase. We summarize a number of experimental techniques to define the accuracy of aminoacylation in vivo and in vitro.
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PMID:The recognition of E. coli glutamine tRNA by glutaminyl-tRNA synthetase. 750 47

The set of nucleotides in Escherichia coli tRNA(Gln) which facilitate aminoacylation by glutaminyl-tRNA synthetase (GlnRS) has been defined [Hayase et al. (1992), EMBO J. 11, 4159-4165]. To determine whether the glutamine "identity set" is sufficient to confer acceptance on a noncognate tRNA, we constructed tRNA(Glu) mutants with the set of glutamine recognition elements. These mutants were examined for aminoacylation in vitro with GlnRS and also with glutamyl-tRNA synthetase (GluRS) to correlate gains in glutamine acceptance with losses of glutamate acceptance. Incorporating glutamine recognition elements in only the acceptor stem or anticodon loop of tRNA(Glu) improved the specificity constant (kcat/KM) for aminoacylation by GlnRS. However, the introduction of all defined glutamine recognition elements in tRNA(Glu) resulted in a substrate with a specificity constant 100-fold below that for aminoacylation of tRNA(Gln). Including the tertiary framework of tRNA(Gln) (in addition to the glutamine recognition elements) in the tRNA(Glu) context further improved aminoacylation by GlnRS, but the specificity was still reduced compared with that of tRNA(Gln). The increase in glutamine acceptance was correlated for all mutants with a decrease in glutamate acceptance, indicating that GluRS also recognizes acceptor stem and anticodon sequences in cognate tRNA. The inability to completely convert tRNA(Glu) to glutamine acceptance with these mutations suggests that tRNA(Glu) contains antideterminants to glutamine identity. The analysis of these mutants with both enzymes revealed that there is a strong element of discrimination between glutamate and glutamine tRNAs associated with the anticodon. To test this dependence, mutants of both tRNAs were made to effect anticodon switches to the possible glutamate and glutamine isoacceptors. The kinetic evaluation of the anticodon switch mutants suggests that overlap in anticodon recognition is avoided through specificity for the third anticodon position coupled with divergent preferences for the wobble base.
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PMID:Discrimination among tRNAs intermediate in glutamate and glutamine acceptor identity. 750 12

The evolution of the aminoacyl-tRNA synthetases is intriguing in light of their elaborate relationship with tRNAs and their significance in the decoding process. Based on sequence motifs and structure determination, these enzymes have been assigned to two classes. The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS), a class I enzyme, complexed to tRNA(Gln) and ATP has been described. It is shown here that a 'minimal' GlnRS, i.e. a GlnRS from which domains interacting with the acceptor-end and the anticodon of the tRNA have been deleted, has enzymatic activity and can charge a tRNA(Tyr)-derived amber suppressor (supF) with glutamine. The catalytic core of GlnRS, which is structurally conserved in other class I synthetases, is therefore sufficient for the aminoacylation of tRNA substrates. Some of these truncated enzymes have lost their ability to discriminate against non-cognate tRNAs, implying a more specific role of the acceptor-end-binding domain in the recognition of tRNAs. Our results indicate that the catalytic and substrate recognition properties are carried by distinct domains of GlnRS, and support the notion that class I aminoacyl-tRNA synthetases evolved from a common ancestor, jointly with tRNAs and the genetic code, by the addition of non-catalytic domains conferring new recognition specificities.
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PMID:Selection of a 'minimal' glutaminyl-tRNA synthetase and the evolution of class I synthetases. 750 22

Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln). Mutations in glnS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection. Two glnS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3.70: glnS113 with an Asp-235-->Asn change selected with GLNA3U70 (GLN carrying G3-->A and C70-->U changes), and glnS114 with a Gln-318-->Arg change selected with GLNU70 (GLN carrying a C70-->U change). The Asp-235-->Asn change was identified previously by genetic selection. Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317-->Arg and Gln-318-->Lys). A number of mutants with no phenotype also were obtained randomly. In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzymes with Lys-317-->Arg, Gln-318-->Lys, or Gln-318-->Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations. However, aminoacylation of a tRNA(Gln) transcript with an opal (UCA) anticodon shows that the specificity constants (kcat/Km) for the mutant enzymes were 5-10 times above that of the wild-type GlnRS. Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS. The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition.
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PMID:Functional communication in the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase. 750 18


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