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

The nucleotides crucial for the specific aminoacylation of yeast tRNA(Asp) by its cognate synthetase have been identified. Steady-state aminoacylation kinetics of unmodified tRNA transcripts indicate that G34, U35, C36, and G73 are important determinants of tRNA(Asp) identity. Mutations at these positions result in a large decrease (19- to 530-fold) of the kinetic specificity constant (ratio of the catalytic rate constant kcat and the Michaelis constant Km) for aspartylation relative to wild-type tRNA(Asp). Mutation to G10-C25 within the D-stem reduced kcat/Km eightfold. This fifth mutation probably indirectly affects the presentation of the highly conserved G10 nucleotide to the synthetase. A yeast tRNA(Phe) was converted into an efficient substrate for aspartyl-tRNA synthetase through introduction of the five identity elements. The identity nucleotides are located in regions of tight interaction between tRNA and synthetase as shown in the crystal structure of the complex and suggest sites of base-specific contacts.
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PMID:Identity elements for specific aminoacylation of yeast tRNA(Asp) by cognate aspartyl-tRNA synthetase. 204 78

We have investigated the functional relationship between nucleotides in yeast tRNAAsp that are important for aspartylation by yeast aspartyl-tRNA synthetase. Transcripts of tRNAAsp with two or more mutations at identity positions G73, G34, U35, C36 and base pair G10-U25 have been prepared and the steady-state kinetics of their aspartylation were measured. Multiple mutations affect the catalytic activities of the synthetase mainly at the level of the catalytic constant, kcat. Kinetic data were expressed as free energy variation at transition state of these multiple mutants and comparison of experimental values with those calculated from results on single mutants defined three types of relationships between the identity nucleotides of this tRNA. Nucleotides located far apart in the three-dimensional structure of the tRNA act cooperatively whereas nucleotides of the anticodon triplet act either additively or anti-cooperatively. These results are related to the specific interactions of functional groups on identity nucleotides with amino acids in the protein as revealed by the crystal structure of the tRNAAsp/aspartyl-tRNA synthetase complex. These relationships between identity nucleotides may play an important role in the biological function of tRNAs.
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PMID:Additive, cooperative and anti-cooperative effects between identity nucleotides of a tRNA. 833 8

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