Showing papers by "Murray P. Deutscher published in 1974"
TL;DR: The results indicate that tRNA nucleotidyltransferase is involved in some aspect of synthesis or repair of the 3' terminus of tRNA, and that the enzyme is present in large excess over its requirements for this function.
Abstract: To determine the function of the enzyme transfer ribonucleic acid (tRNA) nucleotidyltransferase in vivo, five mutants of Escherichia coli containing low levels of this enzyme were isolated. Since no selection procedure for such mutants existed, these strains were isolated by assay of large numbers of colonies from a heavily mutagenized stock. A procedure employing cells made permeable to tRNA and ATP was used to screen the large number of colonies required for the isolation. All the mutants contained less than 20% of the normal level of the AMP-incorporating activity of tRNA nucleotidyltransferase in extracts prepared by several methods, and the best mutant contained only about 2% of this activity. Three of the mutants also had equally low levels of the cytidine 5′-monophosphate-incorporating activity of the enzyme. Despite these low activities, the mutant strains displayed relatively normal growth characteristics at all temperatures examined. The enzyme in the mutant strains was not temperature sensitive, nor were any other abnormal biochemical properties detected. tRNA isolated from the mutant strains was missing significant amounts of its 3′ terminal adenosine 5′-monophosphate residue, amounting to 10 to 15% in the best mutant. However, only small amounts of the terminal cytidine 5′-monophosphate residue were missing. The results indicate that tRNA nucleotidyltransferase is involved in some aspect of synthesis or repair of the 3′ terminus of tRNA, and that the enzyme is present in large excess over its requirements for this function.
55 citations
TL;DR: A series of cca mutants, deficient in the enzyme tRNA nucleotidyltransferase, have been used to show that this activity is essential for the normal growth of Escherichia coli and for the biosynthesis of some tRNAs.
47 citations
TL;DR: Transfer ribonucleic acid (tRNA) nucleotidyltransferase was studied after making cells permeable to macromolecules by treatment with toluene, and the usefulness of such a procedure as an initial step in purification of such enzymes is discussed.
Abstract: Transfer ribonucleic acid (tRNA) nucleotidyltransferase was studied after making cells permeable to macromolecules by treatment with toluene. The conditions of toluene treatment necessary for obtaining maximal activity were defined. Toluene treatment was most efficient when carried out for 5 min at 37 C at pH 9.0 on log-phase cells. No activity could be detected if cells were treated at 0 C, or in the presence of MgCl2, or if the cells were in the stationary phase of growth. However, inclusion of lysozyme and ethylenediaminetetraacetic acid during the toluene treatment did render stationary phase cells permeable. The properties of tRNA nucleotidyltransferase from toluene-treated cells were essentially identical to those of purified enzyme with regard to pH optimum, specificity for nucleoside triphosphates and tRNA, and apparent Km values for substrates. In addition to tRNA nucleotidyltransferase, a variety of other enzymes which incorporate adenosine 5′-triphosphate into acid-precipitable material could also be detected in toluene-treated cells. Centrifugation of cells treated with toluene revealed that tRNA nucleotidyltransferase leaked out of cells, whereas other activities remained associated with the cell pellets. Chromatography of the material extracted from toluene-treated cells on Sephadex G-100 indicated that toluene treatment selectively extracts lower molecular weight proteins. The usefulness of such a procedure as an initial step in purification of such enzymes, and its application to tRNA nucleotidyltransferase, is discussed.
23 citations
TL;DR: It is reported that aminoacyl-tRNA synthetases can be studied within such toluene-treated liver cells, and that these cells are permeable to exogenous tRNA.
21 citations
TL;DR: The proximity of the tolC locus to the integrated sex factor in Hfr strain KL14 may be useful for mapping sex factor mutations by transduction.
Abstract: cca Mutants of Escherichia coli K12 have altered levels of tRNA nucleotidyl-transferase activity. The cca locus has been located at minute 59.4 of the E. coli linkage map. It is cotransduced with tolC but not with argG, and is the earliest known marker transferred by Hfr strain KL14. The proximity of the tolC locus to the integrated sex factor in Hfr strain KL14 may be useful for mapping sex factor mutations by transduction.
21 citations
TL;DR: Examination of specific tRNAs indicated that those specific for isoleucine, leucine and methionine are missing 30 to 40% of their terminal residue, whereas t RNAs specific for tyrosine lack 88% of the 3'-terminal AMP.
Abstract: Essentially all (>97%) of the transfer ribonucleic acid (tRNA) in log-phase and sporulating cells of Bacillus megaterium contains a complete 3′-cytidyl-cytidyl-adenosine terminus. However, about one-third of the tRNA in the dormant spore lacks the 3′-terminal adenosine 5′-monophosphate (AMP) residue, and some of the adjacent cytosine monophosphate residues are also missing. Examination of specific tRNAs indicated that those specific for isoleucine, leucine, and methionine are missing 30 to 40% of their terminal residue, whereas tRNAs specific for tyrosine lack 88% of the 3′-terminal AMP. Defective spore tRNA is not degraded during germination, but the missing residues are added back in the first minutes of the process. The enzyme catalyzing the addition reaction, tRNA nucleotidyltransferase, is present in the dormant spore at a level similar to that found in the vegetative cell.
19 citations
TL;DR: This chapter describes the procedure that leads to the isolation of a high molecular weight complex containing aminoacyl-tRNA synthetases and most of the cellular tRNA, which is extremely unstable and is disrupted by a variety of treatments.
Abstract: Publisher Summary Aminoacyl-tRNA synthetases purified from a variety of sources generally have molecular weights between 70,000 and 200,000 This chapter describes the procedure that leads to the isolation of a high molecular weight complex containing aminoacyl-tRNA synthetases and most of the cellular tRNA Under certain circumstances, only partial complexes are isolated Some of the less tightly bound synthetases are easily removed from the complex The assay measures the conversion of radioactive, acid-soluble amino acids into an acid-insoluble product due to their attachment to tRNA The aminoacyl-tRNA synthetase complex is extremely unstable and is disrupted by a variety of treatments More vigorous homogenization of liver using a motor-driven homogenizer leads to dissociation of the complex The effect of excess homogenization is thought not to be on the complex directly, as it is too small, but is probably due to disruption of other structures, such as lysosomes that could release enzymes that act on components of the complex The process of freezing and thawing destroys the aminoacyl-tRNA synthetase complex
15 citations
TL;DR: This chapter describes the procedures for the purification of the enzyme from Escherichia coli, and rabbit liver, which yields a preparation that is homogeneous and about 30,000-fold purified of tRNA nucleotidyltransferase.
Abstract: Publisher Summary This chapter describes the procedures for the purification of the enzyme from Escherichia coli , and rabbit liver. This procedure yields a preparation of the enzyme from rabbit liver that is homogeneous and about 30,000-fold purified. The assay for tRNA nucleotidyltransferase measures the incorporation of radioactive, acid-soluble adenosine triphosphate (ATP) or cytosine triphosphate (CTP) into an acid-insoluble product. Purified preparations of rabbit liver tRNA nucleotidyltransferase contain a poly(C) polymerase activity as an integral part of the protein. This activity can attach long sequences of cytosine monophosphate (CMP) residues to any intact or partially degraded tRNA molecule or to rRNA. The rate of this activity under the usual assay conditions is only about 1% of the normal CMP incorporation and is generally not detected.
9 citations