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J Bacteriol. 1971 February; 105(2): 527-537
Copyright © 1970 American Society for Microbiology. All Rights Reserved.

Isoleucine Auxotrophy as a Consequence of a Mutationally Altered Isoleucyl-Transfer Ribonucleic Acid Synthetase

International Laboratory of Genetics and Biophysics, Via Marconi 10, 80125 Naples, Italy
Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT

Among mutants which require isoleucine, but not valine, for growth, we have found two distinguishable classes. One is defective in the biosynthetic enzyme threonine deaminase (L-threonine hydro-lyase, deaminating, EC 4.2.1.16) and the other has an altered isoleucyl transfer ribonucleic acid (tRNA) synthetase [L-isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5]. The mutation which affects ileS, the structural gene for isoleucyl-tRNA synthetase, is located between thr and pyrA at 0 min on the map of the Escherichia coli chromosome. This mutationally altered isoleucyl-tRNA synthetase has an apparent K_m for isoleucine (1 mM) 300-fold higher than that of the enzyme from wild type; on the other hand, the apparent V_max is altered only slightly. When the mutationally altered ileS allele was introduced into a strain which overproduces isoleucine, the resulting strain could grow without addition of isoleucine. We conclude that the normal intracellular isoleucine level is not high enough to allow efficient charging to tRNA^Ile by the mutant enzyme because of the K_m defect. A consequence of the alteration in isoleucyl-tRNA synthetase was a fourfold derepression of the enzymes responsible for isoleucine biosynthesis. Thus, a functional isoleucyl-tRNA synthetase is needed for isoleucine to act as a regulator of its own biosynthesis.

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