Specialized lambda transducing bacteriophage which carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase.

A number of specialized lambda transducing bacteriophages which carry the Escherichia coli gene guaB were isolated from E. coli. One of these bacteriophages, lambda cI857 Sam7 d guaB-2, also carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase (EC 6.1.1.21). Histidyl-transfer ribonucleic acid synthetase activities in induced and uninduced lysogens carrying lambda d guaB-2 indicate that the phage carries the entire structural gene and that the gene is under the control of an E. coli promoter. These conclusions were confirmed by the in vivo production of a protein encoded by the phage which comigrates with authentic histidyl-transfer ribonucleic acid synthetase on two-dimensional polyacrylamide gels.     

Genetic analysis of mutations causing borrelidin resistance by overproduction of threonyl-transfer ribonucleic acid synthetase.

Mutations leading to borrelidin resistance in Escherichia coli by overproduction of threonyl-transfer ribonucleic acid synthetase were anaylzed genetically. The regulatory mutations were closely linked to the treonyl-transfer ribonucleic acid synthetase structural gene (thrS), located clockwise to it. The mutation that causes the threefold-increased enzyme level was more distant from thrS than the mutation responsible for the ninefold overproduction. Both mutations were cis dominant in merodiploid strains, indicating that they affected promoter-operator-like control elements. Overproduction was restricted to threonyl-transfer ribonucleic acid synthetase and was not observed for the products of genes neighboring thrS (e.g., infC, pheS, pheT, and argS), providing evidence that thrS is transcribed singly and that gene amplificationis not a likely basis for increased thrS experession.     

Mutants of Escherichia coli Unable to Make Protein at 42 C

Members of a collection of mutants of Escherichia coli unable to form colonies on nutrient agar at 42 C have been characterized on the basis of their growth response to a shift from 32 to 42 C in liquid medium. Forty-four mutants, which show an abrupt, nonlethal cessation of growth when moved to the restrictive temperature, have been characterized with respect to the effect of the mutation responsible for temperature sensitivity on deoxyribonucleic acid, ribonucleic acid, and protein synthesis. In 12 mutants, the mutation causing temperature sensitivity of growth primarily affects protein synthesis, in each case through an altered aminoacyl-transfer ribonucleic acid synthetase. Mutants with temperature-sensitive glutamyl-, phenylalanyl-, and valyl-transfer ribonucleic acid synthetases have been obtained, and the genes specifying these enzymes have been mapped by conjugation and transduction. Another mutant has been shown to possess a temperature-sensitive tryptophanyl-transfer ribonucleic acid synthetase, but this is not responsible for inability to grow at 42 C on media containing tryptophan.     

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.     

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.     

Specificity of translation of spore polypeptides in bacillus in vitro systems

Cell free extracts prepared from exponentially growing Escherichia coli and Bacillus cereus as well as from Bacillus cereus at the end of exponential growth were optimized for various factors required for amino acid incorporation when programmed with Bacillus ribonucleic acid. All three preparations synthesized glutamine synthetase antigen when ribonucleic acid from a Bacillus subtilis strain that overproduces glutamine synthetase was added. The post exponential Bacillus cereus extract, however, was most active for the synthesis of Bacillus cereus spore coat antigen when supplemented with the appropriate ribonucleic acid. There appears to be some specificity in the translation of at least this sporulation messenger RNA.Non-Standard Abbreviations PMSF phenyl methyl sulfonylfluoride - GS glutamine synthetase - UDS 8 M urea, 1% (W/V) sodium dodecyl sulfate, 50 mM dithioerythritol, 2 mM PMSF, 5 mM cyclohexylaminoethane sulfonic acid, pH 9.6     

Evidence for the Existence of Two Arginyl-Transfer Ribonucleic Acid Synthetase Activities in Escherichia coli

Two arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: ribonucleic acid ligase adenosine monophosphate) activities were found in extracts of Escherichia coli strains AB1132 and NP2. The two arginyl-tRNA synthetase activities in extracts of strain AB1132 were found to be separable by diethylaminoethyl-cellulose column chromatography, Sephadex column fractionation, and by sucrose density gradient centrifugation. In addition, in the standard assay using extracts of strain AB1132 there were two pH optima for arginyl-tRNA synthetase activity. Furthermore, when arginyl-tRNA synthetase of strain NP2 was fractionated by hydroxylapatite column chromatography, two activities were observed which were similar to those of strain AB1132.     

Identification of a temperature-sensitive asparaginyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

A temperature-sensitive mutant of Escherichia coli K-12 isolated previously (H. Ohsawa and B. Maruo, J. Bacteriol. 127:1157-1166, 1976) was found to have an alteration in asparaginyl-transfer ribonucleic acid synthetase. This alteration can account for the temperature-sensitive phenotype of the mutant. No evidence was obtained to support the previous suggestion that ribosomal protein S1 is altered in this mutant. Combined with the previous genetic studies, we conclude that the newly defined genetic locus, asnS, for the asparaginyl-transfer ribonucleic acid synthetase maps near pyrD at 21 min on the E. coli chromosome.     

Function of S-adenosylmethionine in germinating yeast ascospores.

Germination and outgrowth of ascospores of Saccharomyces cerevisiae 4579 require both methionine and adenine, whereas leucine is only required for outgrowth. The methionine requirement may be satisfied by S-adenosylmethionine, but this sulfonium compound will not substitute for adenine. Between 30 and 70 min of protein synthesis is initially required for the completion of germination in strain 4579. The inhibition of S-adenosylmethionine synthetase by trifluoromethionine prevents both germination and protein synthesis. During the initial stages of germination, the S-adenosylmethionine synthetase, S-adenosylmethionine decarboxylase, and transfer ribonucleic acid methyltransferases increased significantly, indicating that polyamines and/or the methylation of transfer ribonucleic acid are required for the initiation of germination.     

Transfer Ribonucleic Acid Synthetase Activity Associated with Avian Myeloblastosis Virus

Avian myeloblastosis virions purified by conventional techniques were shown to be associated with or to contain transfer ribonucleic acid synthetase activity. Arginine, tryptophan, cystine, and lysine synthetase activities were observed.     

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.     

Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase

B?ck, 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.     

Isolation and Partial Characterization of Temperature-Sensitive Escherichia coli Mutants with Altered Leucyl- and Seryl-Transfer Ribonucleic Acid Synthetases

Two temperature-sensitive mutants of Escherichia coli have been found in which the conditional growth is a result of a thermosensitive leucyl-transfer ribonucleic acid (tRNA) synthetase and seryl-tRNA synthetase, respectively. The corresponding genetic loci, leuS and serS, cotransduce with lip and serC, respectively. As a result of the mutationally altered leucyl-tRNA synthetase, some leucine-, valine-, and isoleucine-forming enzymes were derepressed. Thus, leucyl-tRNA synthetase is involved in the repression of the enzymes needed for the synthesis of branched-chain amino acids.     

Control of Arginine Biosynthesis in Escherichia coli: Inhibition of Arginyl-Transfer Ribonucleic Acid Synthetase Activity

In this study, we have extended our earlier observations indicating in vitro inhibition of arginyl-transfer ribonucleic acid synthetase (EC 6.1.13, arginine: soluble ribonucleic acid ligase, adenosine monophosphate) activity by the arginine biosynthetic precursors ornithine, citrulline, and argininosuccinate. Furthermore, we report evidence which suggest that this enzyme activity is inhibited by these arginine precursors in vivo and that this inhibition of activity results in a derepression of arginine biosynthesis.     

Mapping of a Structural Gene for Valyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli by Transduction

A structural gene, valS, for the valyl-transfer ribonucleic acid synthetase of Escherichia coli has been mapped on the clockwise side of pyrB and is closely linked to it.     

Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid.     

Role of methionyl-transfer ribonucleic acid in the regulation of methionyl-transfer ribonucleic acid synthetase of Escherichia coli K-12.

A decrease in the in vivo acylation level of methionine transfer ribonucleic acid (tRNAmet) induced by methioninyl adenylate led to a specific derepression of methionyl-transfer ribonucleic acid (tRNA) synthetase formation. This derepression required de novo protein synthesis and was reflected by overproduction of unaltered enzyme. Two different strains of Escherichia coli K-12 that have normal levels of methionyl-tRNA synthetase were examined and the derepression of methionyl-tRNA synthetase was observed in both. Moreover, for one of these strains, the relation between the level of methionyl-tRNA synthetase and deacylation level of tRNAmet was established; under the growth conditions used, when more than 25% of tRNAmet was deacylated, methionyl-tRNA synthetase formation was derepressed and the level of derepression became proportional to the amount of tRNAmet deacylated. Concomitantly, the enzyme was subject to specific inactivation as a consequence of which the true de novo rate of derepression of the formation of this enzyme was higher than that determined by measurements of enzyme activity. These studies were extended to strains AB311 and ed2, which had a constitutive enhanced level of methionyl-tRNA synthetase. In these strains no derepression of enzyme formation was observed on reducing the acylation level of tRNAmet by use of methioninyl adenylate.     

Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. V. Mutants with increased levels of valyl-transfer ribonucleic acid synthetase.

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase were selected for growth at 40 degrees C. Of these, a large number still contain the thermolabile valyl-tRNA synthetase. Three of these revertants contained an increased level of the thermolabile enzyme. The genetic locus, valX, responsible for the enzyme overproduction, is adjacent to the structural gene, valS, of valyl-tRNA synthetase. Determination (by radioimmunoassay) of the turnover rates of valyl-tRNA synthetase showed that the increased level of valyl-tRNA synthetase is due to new enzyme synthesis rather than decreased rates of protein degradation.