CHARACTERIZATION OF A THERMOSENSITIVE ESCHERICHIA-COLI ASPARTYL-TRANSFER-RNA SYNTHETASE MUTANT

The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive g rowth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly cons erved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 stra in, it was shown to restore the wild-type phenotype via homologous rec ombination with the chromosomal tls-1 allele. The mutated AspRS purifi ed from an overproducing strain displayed marked temperature sensitivi ty, with half-life values of 22 and 68 min (at 42 degrees C), respecti vely, for tRNA aminoacylation and ATP/PPi exchange activities. K-m val ues 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 th e amino acid activation sites but has no significant effect on substra te binding. This decrease in stability appears to be related to a conf ormational change, as shown by gel filtration analysis. Structural dat a strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown b y the crystallographic structure of the enzyme, are involved in the ac tive site and in contacts with the tRNA acceptor arm, respectively.