PMID: 9171418Jun 1, 1997Paper

Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant

Journal of Bacteriology
F MartinG Eriani

Abstract

The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the ...Continue Reading

References

Sep 1, 1986·Biochimie·C HountondjiS Blanquet
Jul 19, 1974·Nature·M G RossmannK W Olsen
Jan 1, 1984·Molecular & General Genetics : MGG·C E ShurvintonP V Attfield
Nov 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·G ErianiJ Gangloff
Jan 1, 1996·Acta Crystallographica. Section D, Biological Crystallography·M BoeglinD Moras

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Citations

Nov 6, 2013·Genome Biology and Evolution·Megan WoolfitScott L O'Neill
Aug 26, 2014·Molecular Systems Biology·Matthew ScottTerence Hwa
Jan 5, 2014·The Journal of Microbiology·Kannipa TasanapakGunjana Theeragool
Dec 14, 2016·Nucleic Acids Research·Kamila K MykaPeter McGlynn

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