Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01350 (gastrin)
 9,683 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of wild-type and mutant yeast tRNA(Asp) transcripts with yeast aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) has been probed by using iodine cleavage of phosphorothioate-substituted transcripts. AspRS protects phosphates in the anticodon (G34, U35), D-stem (U25), and acceptor end (G73) that correspond to determinant nucleotides for aspartylation. This protection, as well as that in anticodon stem (C29, U40, G41) and D-stem (U11 to U13), is consistent with direct interaction of AspRS at these phosphates. Other protection, in the variable loop (G45), D-loop (G18, G19), and T-stem and loop (G53, U54, U55), as well as enhanced reactivity at G37, may result from conformational changes of the transcript upon binding to AspRS. Transcripts mutated at determinant positions showed a loss of phosphate protection in the region of the mutation while maintaining the global protection pattern. The ensemble of results suggests that aspartylation specificity arises from both protein-base and protein-phosphate contacts and that different regions of tRNA(Asp) interact independently with AspRS. A mutant transcript of yeast tRNA(Phe) that contains the set of identity nucleotides for specific aspartylation gave a phosphate protection pattern strikingly similar to that of wild-type tRNA(Asp). This confirms that a small number of nucleotides within a different tRNA sequence context can direct specific interaction with synthetase.
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PMID:Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase. 163 Oct 68

Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34.
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PMID:Overproduction and purification of native and queuine-lacking Escherichia coli tRNA(Asp). Role of the wobble base in tRNA(Asp) acylation. 826 43

The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements. It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS. This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems. Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs. It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions. Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T. thermophilus AspRS. In the homologous systems, the mutational effects are less pronounced. However, effects in E. coli and T. thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes. Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex.
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PMID:Identity of prokaryotic and eukaryotic tRNA(Asp) for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus. 865 22

The wild-type transcript of Escherichia coli tRNASec, characterized by a peculiar core architecture and a large variable region, was shown to be aspartylatable by yeast AspRS. Similar activities were found for tRNASec mutants with methionine, leucine, and tryptophan anticodons. The charging efficiency of these molecules was found comparable to that of a minihelix derived from tRNAAsp and is accounted for by the presence of the discriminator residue G73, which is a major aspartate identity determinant. Introducing the aspartate identity elements from the anticodon loop (G34, U35, C36, C38) into tRNASec transforms this molecule into an aspartate acceptor with kinetic properties identical to tRNAAsp. Expression of the aspartate identity set in tRNASec is independent of the size of its variable region. The functional study was completed by footprinting experiments with four different nucleases as structural probes. Protection patterns by AspRS of transplanted tRNASec and tRNAAsp were found similar. They are modified, particularly in the anticodon loop, upon changing the aspartate anticodon into that of methionine. Altogether, it appears that recognition of a tRNA by AspRS is more governed by the presence of the aspartate identity set than by the structural framework that carries this set.
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PMID:The peculiar architectural framework of tRNASec is fully recognized by yeast AspRS. 1019 66

The active site of yeast aspartyl-tRNA synthetase has been characterised by structural and functional approaches. However, residues or structural elements that indirectly contribute to the active site organisation have still to be described. They have not been assessed by simple analysis of structural data or site-directed mutagenesis analysis, since rational targetting has proven difficult. Here, we attempt to locate these functional features by using a genetic selection method to screen a randomly mutated yeast AspRS library for mutations lethal for cell growth. This approach is an efficient method to map the active site residues, since of the 23 different mutations isolated, 13 are in direct contact with the substrates. Most of the mutations are located in a 15 A radius sphere around the ATP molecule, where they affect the very conserved residues of the class-defining motifs. The results also showed the importance of the dimer interface for the enzyme activity: a single mutation of the invariant proline residue of motif 1 led to a structural defect inactivating the enzyme. From in vivo complementation studies it appeared that the enzyme activity can be recovered by reconstitution of an intact interface through the formation of heterodimers. We also show that a single mutation affecting an interaction with G34 of the tRNA can inactivate the enzyme by inducing a relaxation of the tRNA recognition specificity. Finally, several mutants whose functional importance could not be assessed from the structural data were selected, demonstrating the importance of this type of approach in the context of a structure-function relationship study.
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PMID:Active site mapping of yeast aspartyl-tRNA synthetase by in vivo selection of enzyme mutations lethal for cell growth. 1032 39