tRNA nucleotidyltransferase is not essential for Escherichia coli viability.

The role of tRNA nucleotidyltransferase in Escherichia coli has been uncertain because all tRNA genes studied in this organism already encode the -C-C-A sequence. Examination of a cca mutant, originally thought to contain 1-2% enzyme activity, indicated that it actually produces an inactive fragment of 40 kd compared to 47 kd for the wild-type enzyme due to a nonsense mutation in its cca gene. To confirm that the residual activity in extracts of this strain is due to another enzyme, and that tRNA nucleotidyltransferase is non-essential, we have interrupted the cca gene in vitro, and transferred this mutant gene to a variety of strains. In all cases mutant strains are viable, although as much as 15% of the tRNA population contains defective 3' termini, and no tRNA nucleotidyltransferase is detectable. Mutant strains grow slowly, but can be restored to more normal growth by a relA mutation or by a decrease in RNase T activity. In the latter case the amount of defective tRNA decreases dramatically. These findings indicate that tRNA nucleotidyltransferase is not essential for E. coli viability, and therefore, that all essential tRNA genes in this organism encode the -C-C-A sequence.     

Expression of double-stranded-RNA-specific RNase III of Escherichia coli is lethal to Saccharomyces cerevisiae.

The gene for the double-stranded RNA (dsRNA)-specific RNase III of Escherichia coli was expressed in Saccharomyces cerevisiae to examine the effects of this RNase activity on the yeast. Induction of the RNase III gene was found to cause abnormal cell morphology and cell death. Whereas double-stranded killer RNA is degraded by RNase III in vitro, killer RNA, rRNA, and some mRNAs were found to be stable in vivo after induction of RNase III. Variants selected for resistance to RNase III induction were isolated at a frequency of 4 X 10(-5) to 5 X 10(-5). Ten percent of these resistant strains had concomitantly lost the capacity to produce killer toxin and M dsRNA while retaining L dsRNA. The genetic alteration leading to RNase resistance was localized within the RNase III-coding region but not in the yeast chromosome. These results indicate that S. cerevisiae contains some essential RNA which is susceptible to E. coli RNase III.     

On the binding of tRNA to Escherichia coli RNA polymerase.

The fixation of tRNA to Escherichia coli RNA polymerase has been investigated. Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels. We detect no differences between the fixation of E. coli fMet-tRNAfMet, Met-tRNAmMet or uncharged unfractionated tRNA to RNA polymerase. Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and both monomer and dimer forms of the core enzyme. In the monomer complex, one tRNA is bound per alpha 2 beta beta' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per alpha 2 beta beta' unit. In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly. DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist. Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase.     

Mammalian prothymosin alpha links to tRNA in Escherichia coli cells.

Prothymosin alpha, a small and highly acidic nuclear protein related to cell proliferation, is known to be covalently attached to a small unidentified cytoplasmic RNA in mammalian cells. Here we demonstrate that recombinant rat prothymosin a links covalently to an RNA when overproduced in Escherichia coli cells. The RNA species of this complex is represented by a wide range of bacterial tRNAs. tRNA(Lys), tRNA(3Ser), tRNA(2Ile), and tRNA(mMet) were identified by sequencing. Prothymosin alpha appears to be linked to the 5' terminus of tRNA. tRNA attachment site lies close to the carboxy-terminus of prothymosin alpha. Furthermore, the carboxy-terminal peptide of prothymosin alpha is also competent for tRNA binding. The site of tRNA attachment coincides with the nuclear localization signal of prothymosin alpha, and tRNA binding might be expected to affect subcellular localization of this protein.     

Reinvestigation of phosphorylation of tRNA in Escherichia coli.

This report shows the results of the reinvestigation of tRNA phosphorylation in E. coli. The phosphorylation did not occur on suppressor seryl-tRNA but occurred on other tRNA species. The activity of tRNA phosphorylation was found in E. coli extracts and partially purified. On DEAE-Sephadex A50 and PAGE gel, the phosphorylated-tRNA showed a pattern different from that the natural suppressor serine tRNA.     

Kinetic mechanism of tRNA nucleotidyltransferase from Escherichia coli.

A kinetic analysis of the incorporation of AMP into tRNA lacking the 3'-terminal residue by tRNA nucleotidyltransferase (EC 2.2.7.25) from Escherichia coli is presented. Initial velocity studies demonstrate that the mechanism is sequential and that high concentrations of tRNA give rise to substrate inhibition which is noncompetitive with respect to ATP. In addition, the substrate inhibition is more pronounced in the presence of pyrophosphate, which suggests the formation of an inhibitory enzyme-pyrophosphate-tRNA complex. Noncompetitive product inhibition is observed between all possible pairs of substrates and products. ADP and alpha,beta-methylene adenosine triphosphate are competitive dead end inhibitors of ATP, while the latter is a noncompetitive dead end inhibitor of the tRNA substrate. A nonrapid equilibrium random mechanism is proposed which is consistent with these data and offers an explanation for the noncompetitive substrate inhibition by tRNA.     

Affinity labeling of the ribonucleic acid component adjacent to the peptidyl recognition center of peptidyl transferase in Escherichia coli ribosomes.

N-Iodacetylphenylalanyl-tRNA was used as an affinity label for localizing the RNA components intimately related to the peptidyl transferase activity of Escherichia coli ribosomesmthis analogue could specifically alkylate a unique nucleotide chain of 23-S RNA. The alkylation was strongly enhanced by poly(U), and was dependent on the presence of both 50- and 30-S subunits; Chloramphenicol inhibited the reaction, wheras blasticidin S stimulated it. The alkylated RNA base was found to be adenine. The nucleotide chain attacked by N-iodoacetylphenylalanyl-tRNA seemed to be localized at or near to the peptidyl recognition center of peptidyl transferase.     

Accumulation of glutamate by osmotically stressed Escherichia coli is dependent on pH.

In the present study, we measured the accumulation of glutamate after hyperosmotic shock in Escherichia coli growing in synthetic medium. The accumulation was high in the medium containing sucrose at a pH above 8 and decreased with decreases in the medium pH. The same results were obtained when the hyperosmotic shock was carried out with sodium chloride. The internal level of potassium ions in cells growing at a high pH was higher than that in cells growing in a neutral medium. A mutant deficient in transport systems for potassium ions accumulated glutamate upon hyperosmotic stress at a high pH without a significant increase in the internal level of potassium ions. When the medium osmolarity was moderate at a pH below 8, E. coli accumulated gamma-aminobutyrate and the accumulation of glutamate was low. These data suggest that E. coli uses different osmolytes for hyperosmotic adaptation at different environmental pHs.     

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.     

The nucleotide sequence of asparagine tRNA from Escherichia coli.

The nucleotide seuquence of Escherichia coli asparagine tRNA was determined to be pU-C-C-U-C-U-G-s4U-A-G-U-U-C-A-G-D-C-G-G-D-A-G-A-A-C-G-G-C-G-G-A-C-U-Q-U-U-t6A-A-phi-C-C-G-U-A-U-m G-U-C-A-C-U-G-G-T-phi-C-G-A-G-U-C-C-A-G-U-C-A-G-A-G-G-A-G-C-C-AOH. Its D-stem and D-loop have almost the same sequence as Escherichia coli aspartate tRNA.     

Purification and some properties of Escherichia coli tRNA nucleotidyltransferase.

Adenylyl (cytidylyl)-tRNA nucleotidyltransferase (ATP (CTP): tRNA adenylyl (cytidylyl)transferase, EC2.7.7.25) has been purified 11,800-fold from a crude extract of Escherichia coli B in an overall yield of 23%. The key step in this purification is the use of a tRNA-Sepharose affinity column. The purified enzyme has a specific activity of approximately 280 mumol of AMP incorporated/min/mg of protein at 37 degrees and has a molecular weight of 52,000 as determined by sodium dodecyl sulfate gel electrophoresis of Sephadex chromatography. The turnover number of the pure enzyme, under optimal assay conditions, is estimated as 21,000, and we believe it constitutes only o.oo6% of the total cellular protein. Both AMP- and CMP-incorporating activities have an identical isoelectric point of 5.85. The AMP-incorporating activity of the enzyme is inhibitied by some transition metal chelating agents but not by others.     

RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells.

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.     

Expression of gene 1.2 and gene 10 of bacteriophage T7 is lethal to F plasmid-containing Escherichia coli.

Plasmids expressing bacteriophage T7 gene 1.2 or gene 10 DNA transform F plasmid-containing strains of Escherichia coli only at low efficiency, though they transform plasmid-free strains normally. The gene products T7 gp1.2 and T7 gp10 appear to be the toxic agents, and their effects are directed towards the product of the F pifA gene, PifA. T7 gp1.2 and gp10 are also the two targets of the pif exclusion system of F, and their synthesis normally triggers the abortive infection of T7 in pifA+ hosts. The properties of plasmids containing T7 gene 1.2 or 10 suggest that they can be used to study the molecular mechanisms of phage exclusion in model systems that avoid the pleiotropic dysfunctions associated with an abortive infection.     

The isolation of a 4-thiouridine-containing disulfide from oxidized Escherichia coli tRNA is not artifactual.

Iodine-oxidized Escherichia coli tRNA yields an oxidized 4-thiouridine dinucleoside after enzymatic digestion and dephosphorylation. The original isolation has been questioned on the basis of an observed instability of 4-thiouridine disulfide in Tris-Cl buffer and a persistent oxidizing capacity of some resins after exposure to iodine. It is here pointed out that the original isolation specifically negated both of these objections. The fragment recently isolated by Kaiser (Arch. Biochem. Biophys.183, 421–431, 1977) appears to be very similar to the one previously reported, although probably neither one is the symmetrical 4-thiouridine disulfide.     

Comparison of tRNA nucleotidyltransferase from Escherichia coli and Lactobacillus acidophilus.

Purification of tRNa nucleotidyltransferase from Lactobacillus acidophilus ATCC 4963 and Escherichia coli MRE 600 by preparative polyacrylamide gel electrophoresis is described. Both enzymes gave a single band on analytical polyacrylamide-gel electroesis and sodium dodecylsulfate gels. Chromatography of the high speed supernatant from Lactobacillus at low salt concentrations gave three enzyme fractions of molecular weights about 45 000, 90 000, and 120 000. At 1M NaCl only the first enzyme fraction was found. Kinetic data for both enzymes are given.     

Accumulation of Tetracyclines by Escherichia coli

The net accumulation of tetracyclines by Escherichia coli as a function of concentration was shown to be biphasic. At concentrations less than the bacteriostatic levels, the mode of uptake was not azide-sensitive and was considered to be physical adsorption on the cell surface. At concentrations above the minimal inhibitory level, a second, azide-sensitive, uptake component was functional in addition to the surface adsorption process. This second energy-requiring mode was judged to represent penetration of the cytoplasmic membrane by tetracycline molecules to their sites of inhibitory action. Each mode for a given tetracycline and culture is expressed algebraically by a characteristic Freundlich equation. Resistance in E. coli is shown to be a result of diminished transport of antibiotic. However, this resistance was due not to a reduction or loss of a transport mechanism but rather to a requirement for higher antibiotic concentrations before the second mode of uptake could become operative.