PHYSIOLOGICAL ROLES OF AMINO ACID ACTIVATION IN BACTERIA

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Protein and Nucleic Acid Synthesis in Two Mutants of <i>Escherichia coli</i> with Temperature-Sensitive Aminoacyl Ribonucleic Acid Synthetases

Eidlic, Lia (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt . Protein and nucleic acid synthesis in two mutants of Escherichia coli with temperature-sensitive aminoacyl ribonucleic acid synthetases. J. Bacteriol. 89: 706–711. 1965.—Two temperature-sensitive mutants of Escherichia coli were isolated which grow almost normally at 30 C and fail to grow at 37 C. One (I-9) was derived from a strain with stringent amino acid control of ribonucleic acid (RNA) synthesis; the other (IV-4) was derived from a strain with relaxed amino acid control of RNA synthesis. When cultures of these mutants growing at 30 C were shifted to 37 C, IV-4 synthesized RNA preferentially to protein but I-9 did not. Cell-free extracts of both mutants and their parent strains were examined for their ability to catalyze adenosine triphosphate (ATP)-dependent attachment of amino acids to soluble RNA (sRNA). These measurements indicated that I-9 possesses a temperature-sensitive valyl sRNA synthetase, and that IV-4 possesses a temperature-sensitive phenylalanyl sRNA synthetase. The behavior of these mutants suggests that amino acids permit RNA synthesis in stringent strains only after activation or attachment to sRNA, that relaxed strains can overproduce RNA without a complete array of fully functioning aminoacyl sRNA synthetases, and that these enzymes are obligatory for the biosynthesis of proteins. </jats:p

Biochemical and Genetic Characterization of a Mutant of <i>Escherichia coli</i> with a Temperature-Sensitive Valyl Ribonucleic Acid Synthetase

Böck, August (Purdue University, Lafayette, Ind.), Lia Eidlic Faiman, and Frederick C. Neidhardt . Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase. J. Bacteriol. 92: 1076–1082. 1966.—To test our conclusion that Escherichia coli mutant I-9 possesses a valyl soluble ribonucleic acid (sRNA) synthetase that functions in vivo at 30 C but not at 37 C, measurements were made by use of the periodate method, of the level of charged valyl sRNA in this strain. A shift of temperature from 30 to 40 C resulted in a rapid discharging of valyl sRNA coordinate with the cessation of protein synthesis; at the same time, other species of sRNA, such as those for leucine, became fully charged. Identical results were obtained with a derivative of I-9 with relaxed ribonucleic acid (RNA) control. When P1 phage were grown on wild cells and then used at low multiplicities of infection to transduce temperature-resistant growth into I-9, complete cotransduction of normal valyl sRNA synthetase occurred. By means of the interrupted-mating technique, the structural gene for valyl sRNA synthetase was located on the E. coli chromosome map and found to be near thr , one-fifth of the length of the chromosome removed from the structural genes for the isoleucine-valine biosynthetic enzymes. Therefore, (i) the major valyl sRNA synthetase activity of I-9 appears to be temperature-sensitive in vivo, (ii) relaxed amino acid control over RNA synthesis does not appear to be a consequence of a normal charging of sRNA with a substitute molecule, and (iii) one structural gene for valyl sRNA synthetase is located on the E. coli chromosome not closely linked to the cistrons for the valine-biosynthetic enzymes. </jats:p