Gene organization around the phenylalanyl-transfer ribonucleic acid synthetase locus in Escherichia coli.

The organization of seven genes located at about 38 min on the genetic map of Escherichia coli was examined; these genes included pheS and pheT, which code for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid synthetase, and thrS, the structural gene for threonyl-transfer ribonucleic acid synthetase. Deletion mutants were isolated from an F-prime-containing merodiploid strain and were characterized genetically. Seventeen different kinds of deletions extending into pheS of pheT were identified. These deletions unambiguously defined the gene order as aroD pps himA pheT pheS thrS pfkB. Mutants with deletions covering either pheS or pheT, but not both, were analyzed further by assay of phenylalanyl-transfer ribonucleic acid synthetase. The phenotype of the mutants with a deletion from pfkB through pheS was anomalous; although the pheT gene was apparently still present, its product, the beta subunit, was much reduced in activity.     

Genes for the alpha and beta subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli.

The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains. To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subunit was altered. The method was in vitro complementation; mutant cell extracts were mixed with purified separated alpha or beta subunits of the wild-type enzyme to generate an active hybrid enzyme. With three mutants, enzyme activity appeared when alpha was added, but not when beta was added: these are, therefore, assumed to carry lesions in the gene for the alpha subunit. Two other mutants gave the opposite response and are presumably beta mutants. Enzyme activity is also generated when alpha and beta mutant extracts are mixed, but not when two alpha or two beta mutant extracts are mixed. The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial. The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly. Both alpha and beta mutants are capable of generating the native form, although alpha mutants require prior urea denaturation of the defective enzyme. With the mutants thus characterized, the genes for the alpha and beta subunits (designated pheS and heT, respectively) were mapped. The gene order, as determined by transduction is aroD-pps-pheT-pheS. The pheS and pheT genes are close together and may be immediately adjacent.     

Pleiotropic phenotype of an Escherichia coli mutant lacking leucyl-, phenylalanyl-transfer ribonucleic acid-protein transferase.

A mutant of Escherichia coli that lacks leucyl-, phenylalanyl-transfer ribonucleic acid-protein transferase had diminished activities of L-phenylalanyl-transfer ribonucleic acid synthetase and tryptophanase, grew faster than its parent with aspartic acid as the sole nitrogen source, accumulated higher levels of enterochelin in the medium during iron limitation, and exhibited an abnormal morphology.     

Cross-reactivity of phenylalanyl-transfer ribonucleic acid ligases from different microorganisms.

The cross-reaction of phenylalanyl-transfer ribonucleic acid (tRNA) ligases from different microorganisms with antibodies raised against the purified enzyme from Escherichia coli has been investigated. The results of immunotitration and immunodiffusion experiments and of the sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of immunoprecipitates revealed: (i) a high degree of immunochemical identity of this enzyme only within the family Enterobacteriaceae; (ii) intermediate-to-weak cross-reaction with the phenylalanyl-tRNA ligases from Pseudomonadaceae, Rhodopseudomonas spheroides, and Bacillus stearothermophilus; (iii) no detectable cross-reaction (with the methods employed) with the enzymes from several gram-positive organisms, Euglena gracilis, and several fungi. As revealed by immunochemical analysis, a merodiploid strain of E. coli carrying an episome (F148) that covers the aroD region of the E. coli chromosome possesses at least twice the amount of phenylalanyl-tRNA ligase in comparison with its haploid parent strain. This suggests that the cistrons for both the alpha and beta polypeptides of this enzyme are mapping in this area.     

The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase.

The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.     

Deletion of a ribosomal ribonucleic acid operon in Escherichia coli.

One (rrnE) of the seven operons which codes for ribosomal ribonucleic acid in Escherichia coli was deleted. No significant change in phenotype was observed even under maximum laboratory growth conditions.     

Multiple forms of lysyl-transfer ribonucleic acid synthetase in Escherichia coli.

Lysyl-transfer ribonucleic acid synthetase (EC 6.1.1.6) was identified as four polypeptide spots after two-dimensional polyacrylamide gel electrophoresis of whole-cell lysates of Escherichia coli. Identification was made by migration with partially purified enzyme preparations, by peptide map patterns, by mutant analysis, and by correlation of spot intensities with changes in enzyme levels under different growth conditions. Wild-type cells growing at 37 degrees C in glucose minimal medium displayed the enzyme predominantly as two spots (spots I and III). Growth at 46 degrees C, growth in the presence of alanine or glycyl-L-leucine, or growth of a strain with a mutational deficiency in S-adenosylmethionine synthetase (metK) greatly increased the synthesis of two other spots (spots II and IV). Polypeptides I and III, but not polypeptides II and IV, had altered isoelectric points in a lysyl-transfer ribonucleic acid synthetase mutant. These data suggest that multiple forms of lysyl-transfer ribonucleic acid synthetase exist in vivo and that they may be encoded by more than one gene.     

Galactose-specific messenger ribonucleic acid contents in Escherichia coli

A method is described for measuring the proportion of galactose-specific mRNA (gal-mRNA) in the total RNA extracted from pulse-labelled cells of Escherichia coli K12, by DNA-RNA hybridization with DNA prepared from bacteriophage lambdadg. RNA from wild-type E. coli was compared with RNA from a homogenote carrying the gal operon both in the chromosome and in a substituted sex-factor, and with RNA from a deletion strain that carried the galactose operon only in the exogenote. In each case the cultures were induced with fucose. Under these conditions the amount of gal-mRNA was found to be proportional to the content of galactokinase in the different cultures, and to the gene frequency. The amounts of gal-mRNA in an O(c) mutant and an R(-) mutant were also proportional to the observed contents of galactokinase. In cultures repressed for the enzymes of the galactose operon with thiomethylgalactoside, the content of gal-mRNA was higher than expected from the content of galactokinase. Possible explanations of this finding are discussed.