Polypeptide Synthesis by Extracts from Escherichia coli Treated with T2 Ghosts

Infection of Escherichia coli B in inorganic salts-glycerol with a multiplicity of deoxyribonucleic acid-less T2 "ghosts" just sufficient to block all protein synthesis results in both viable and killed bacteria. We enriched for the viable cells by a combination of lysozyme treatment and filtration and measured the in vitro capacity of their extracts to synthesize polypeptides. Without added template ribonucleic acid (RNA), such "ghost extracts" incorporate amino acids (endogenous synthesis) at approximately one-half the rate as do extracts from uninfected bacteria. However, they are unable to use added synthetic or natural template RNAs for peptide synthesis. Some activity can be observed but only at high concentrations of Mg(2+). These results suggest that ghost infection may result in a blockage of ribosomes during translation. Mixing experiments show that the incapacity of ghost extracts to translate added template RNA is due to a defect in the ribosomes.     

The effect of ribonuclease on polysomes and ribosomes of bacteria and animal cells

The mildest treatment with ribonuclease that causes any disaggregation of the polysomes of Escherichia coli or HeLa cells simultaneously attacks the RNA of the constituent ribosomes. It is concluded that the susceptibility to ribonuclease of polysomes does not suggest that they are held together by a strand of messenger RNA. The RNA of the larger sub-unit of bacterial ribosomes has particularly sensitive regions resulting in a non-random degradation. The RNA of the smaller sub-unit of E. coli ribosomes is relatively resistant to ribonuclease attack. The same may be true of the respective sub-units of the intact HeLa-cell ribosome, but both sub-units become very sensitive to ribonuclease on dissociation from each other.     

Streptomycin Action: Greater Inhibition of Escherichia coli Ribosome Function with Exogenous than with Endogenous Messenger Ribonucleic Acid

Inhibition of protein synthesis by streptomycin was tested in extracts from a strain of Escherichia coli sensitive to streptomycin. Three kinds of messenger ribonucleic acid (RNA) were employed: endogenous cellular RNA, extracted cellular RNA, and phage R17 RNA. Protein synthesis directed by extracted cellular RNA was inhibited three- to fourfold more than protein synthesis directed by endogenous RNA. With R17 RNA as messenger, nearly total inhibition of protein synthesis at initiation was again observed. The greater inhibition of function of extracted RNA, which must initiate new polypeptide chains in vitro, is in accord with the observation that in whole cells streptomycin blocks ribosomes at an early stage in protein synthesis. When streptomycin was added at successively later times during protein synthesis, the subsequent inhibition was progressively less. This was observed with either extracted cellular RNA or phage R17 RNA. A model is presented that can explain the less drastic inhibition by streptomycin of messenger RNA that is already functioning on ribosomes.     

Altered ribosomes after inhibition of Escherichia coli by rifampicin

Addition of rifampicin to growing cells of Escherichia coli affected the ribosomes. The polyribosomes first decayed to 70S ribosomes. These later dissociated to particles distinct from ribosomal subunits. The altered ribosomes sedimented more slowly than the corresponding subunits and had lost some protein; their ribosomal RNA was intact, but they were more susceptible to degradation by ribonuclease than normal ribosomes. The addition of rifampicin to preparations of lysed cells caused no detectable changes in the ribosome fraction.     

Role of ribosome degradation in the death of starved Escherichia coli cells.

In Escherichia coli cultures limited for phosphate, the number of ribosomal particles was reduced to a small percentage of its earlier peak value by the time the viable cell count began to drop; the 30S subunits decreased more than the 50S subunits. Moreover, the ribosomal activity was reduced even more: these cells no longer synthesized protein, and their extracts could not translate phage RNA unless ribosomes were added. The translation initiation factors also disappeared, suggesting that they become less stable when released from their normal attachment to 30S subunits. In contrast, elongation factors, aminoacyl-tRNA synthetases, and tRNA persisted. During further incubation, until viability was reduced to 10(-5), the ribosomal particles disappeared altogether, while tRNA continued to be preserved. These results suggest that an excessive loss of ribosomes (and of initiation factors) may be a major cause of cell death during prolonged phosphate starvation.     

Ribonuclease sensitivity of Escherichia coli ribosomes

Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099-1110. 1966.-The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10(-4)m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities.     

Decomposition of ribosomal particles in Escherichia coli treated with mitomycin C

Exposure of cells of Escherichia coli to mitomycin C (5 mug/ml) resulted in a marked change in the sedimentation profiles of the cell-free extracts, indicating a specific decomposition of ribosomal particles. When the extracts were prepared in the presence of 0.01 m Mg(++) and analyzed by sucrose density gradient centrifugations, the 100S fraction disappeared rapidly from the treated cells. The 70S ribosomes were also degraded, but more slowly, with a concomitant accumulation of a fraction having a sedimentation coefficient of about 50S. However, decomposition of the 70S ribosomes was preceded by an almost complete loss of the 50S ribosomal subunits, as revealed by sedimentation analyses in the presence of 10(-4)m Mg(++). Synthesis of the ribosomes in the treated cells was also suppressed, being demonstrated by a lower incorporation of uracil-2-(14)C into the ribosomal fractions. However, the change in the ribosomal profile in the treated cells apparently resulted from the decomposition of pre-existing ribosomes, rather than from the inhibition of the net synthesis of ribosomes. Sedimentation analyses and chromatography of the nucleic acids extracted from the treated cells indicated extensive but delayed degradation of the ribosomal ribonucleic acid (RNA), but not of the soluble RNA or deoxyribonucleic acid fractions. Altered structure of the ribosomes in the treated cells was also indicated by their lower melting temperature, broadened thermal profile, higher electrophoretic mobility, and extreme sensitivity to ribonuclease treatment, compared with normal ribosomes. The synthesis of messenger RNA was inhibited progressively with time in the treated cells.     

Functional modifications of the translational system in Bacillus subtilis during sporulation.

Extracts of sporulating cells were found to be defective in vitro translation of phage SP01 ribonucleic acid (RNA) and vegetative Bacillus subtilis RNA. The activity of washed ribosomes from sporulating cells was very similar to that of washed ribosomes from vegetative cells in translating polyuridylic acid, SP01 RNA, and vegetative RNA. The S-150 fraction from either vegetative or sporulating cells grown in Difco sporulation medium contained an apparent inhibitor of protein synthesis. The crude initiation factor fraction from ribosomes of sporulating cells was defective in promoting the initiation factor-dependent translation of SP01 RNA. The crude initiation factor preparations from sporulating cells were as active as the corresponding preparations from vegetative cells in promoting the initiation factor-dependent translation of either phage Qbeta or phage T4 RNA by washed Escherichia coli ribosomes. The crude initiation factors from sporulating cells were perhaps more active than those from vegetative cells in promoting the initiation factor-dependent synthesis of phage T4 lysozyme by E. coli ribosomes. The crude initiation factor preparations from either vegetative or stationary-phase cells of an asporogenous mutant showed similar ability to promote the in vitro translation of SP01 RNA.     

Isolation of a Mutant of Escherichia coli with a Temperature-sensitive Fructose-1,6-Diphosphate Aldolase Activity

B?ck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Isolation of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase activity. J. Bacteriol. 92:464-469. 1966.-A mutant of Escherichia coli was isolated which was able to grow in rich medium at 30 C but not at 40 C. Upon exposure to 40 C, the cells immediately stopped ribonucleic acid (RNA) and deoxyribonucleic acid synthesis, but protein synthesis continued at a diminished rate for a short time. Addition of chloramphenicol did not release RNA synthesis from inhibition at 40 C. Synthesis of beta-galactosidase could be induced at high temperature despite the presence of glucose in the medium, indicating a lesion in glucose catabolism. Of many catabolic enzymes tested in cell-free extracts, only fructose-1,6-diphosphate aldolase activity appeared to be altered in the mutant cells. No activity was demonstrable in extracts of mutant cells grown at either 30 or 40 C, but determination of glucose-oxidation patterns revealed that the enzyme is probably active in vivo at 30 C. Temperature-resistant secondary mutants were found to have partially or fully restored aldolase activity, and temperature-resistant recombinants had normal aldolase activity, indicating that the growth pattern and the altered aldolase had a common genetic basis. Linkage data permitted the assignment of an approximate map location for the mutated aldolase gene.     

In vivo incorporation of the intrinsic photolabel 4-thiouridine into Escherichia coli RNAs.

The in vivo incorporation of the photoactivable uridine analogue 4-thiouridine into the RNAs of an Escherichia coli K12 pyrD strain has been optimised. s4Urd uptake in RNAs appears to be strikingly dependent upon the age of the preculture, i.e. the number of generations the cells have undergone immediately before dilution in the thiolation medium. Conditions have been set up where efficient RNA thiolation occurs in cells growing exponentially at 50 to 70% the rate of the control. The substitution level s4U/U is maximal after growth for 9 to 10 generations in the thiolation medium and reaches 17 +/- 3% in tRNA and bulk RNA. Most of ribosomal derived ribonucleoproteins, 65 +/- 5%, sediment as 70S ribosomes (s4U/U = 7 +/- 2%) on a high Mg2+ sucrose gradient. The thiolated RNAs were characterized by their migration on a thiol-specific affinity electrophoretic gel.     

Structural Transitions in Ribonucleic Acid During Ribosome Development

Amino acid deprivation of a "relaxed" auxotroph of Escherichia coli results in the accumulation of protein-deficient, immature ribosomes ("relaxed particles"). The ribonucleic acid (RNA) of these particles was shown to differ from mature ribosomal RNA in both sedimentation characteristics and in elution from columns of methylated albumin-keiselguhr. When relaxed particles were allowed to become converted to mature ribosomes, the unique properties of the RNA were lost, and this RNA became indistinguishable from mature RNA. The conversion of relaxed particles to ribosomes did not involve degradation and resynthesis of RNA. It is concluded that ribosomal RNA undergoes a configurational transition during ribosome development, and that this transition is not the result of changes in the primary structure of the RNA.     

Translation of Virus mRNA: Synthesis of Bacteriophage Qβ Proteins in a Cell-Free Extract from Wheat Embryo

The RNA from bacteriophage Qbeta can be translated by cell-free extracts from wheat embryos. This translation, by 80S ribosomes, occurs at a low magnesium ion concentration. Three products are synthesized which coelectrophorese with Qbeta proteins synthesized in Escherichia coli extracts. The smallest of these has been identified as coat protein. Although the polycistronic bacteriophage message is translated with fidelity, the efficiency is much less than when the monocistronic brome mosaic virus coat protein message is translated.     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB+ gene of the F plasmid, rifampin, added at 42 degrees C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257-258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784-789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolydigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (Mr 12 000) newly synthesized at 42 degrees C in the presence of rifampin appeared to be the product of the srnB+ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30 degrees C, or the addition of Mg2+ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB+ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB+ gene.     

Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system. J. Bacteriol. 91:1917-1923. 1966.-Tetracycline, an inhibitor of cell-free protein synthesis, effected the dissociation of Escherichia coli 100S ribosomes to 70S particles in vivo and in vitro, but was not observed to mediate the further degradation of these particles. The antibiotic was bound by both 50S (Svedberg) and 30S subunits of 70S ribosomes and also by E. coli soluble RNA (sRNA), polyuridylic acid (poly U), and polyadenylic acid (poly A). The binding to ribosomal subunits was higher at 5 x 10(-4)m Mg(++) than at 10(-2)m Mg(++). The binding to polynucleotide chains was highest when Mg(++) was not added to the reaction mixture.     

Morphological and physiological changes induced by high hydrostatic pressure in exponential- and stationary-phase cells of Escherichia coli: relationship with cell death

The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.     

Homologous RNA polymerase binding to Escherichia coli DNA: a study of the distribution of stable binding sites.

The number and the distribution of the sites of Escherichia coli DNA that form stable complexes with the homologous RNA polymerase (class A sites according to Hinkle and Chamberlin [3]) have been investigated. Almost all the DNA can bind RNA polymerase, even when fragmented at short (undergenic) size; this general non-promoter-specific binding is highly labile and is not temperature-dependent. The range of RNA polymerase/DNA ratios that give rise to the stable temperature-dependent complexes was examined. The amount and the distribution of class A complexes were studied analysing the dissociation of complexes formed by RNA polymerase on DNA fragments of various length. The E. coli genome appears to form 3.8 X 10(3) stable complexes; the majority of these complexes shows a short-range distribution (800-1200 base pairs). The rest is more widely spaced (1200-6000 base pairs).     

Polypeptide synthesis in toluene-treated lymphocytes

Normal human lymphocytes stimulated by phytohemagglutinin P, and other mammalian cells were rendered permeable to macromolecules such as poly(U) and proteins, by treatment with a low concentration of toluene. Under this condition, poly(U) translation was more efficient in the permeabilized cells than in 10000 X g extracts. Such a process occurs inside the treated cells as demonstrated by the fact that [14C]uridine-labelled ribosomes remain associated with the toluene-treated lymphocytes even after incubation at 37 degrees C. A nuclease from Staphylococcus aureus was able to penetrate the permeabilized cells and to break the polysome-bound endogenous messenger RNA. However, the protein-synthesizing machinery inside the toluene-treated lymphocytes was unaffected by the nuclease, as demonstrated by the unimpairment of polyphenylalanine synthesis when poly(U) was added after the preincubation with the enzyme. These results suggest that the toluene treatment can be considered as an important tool for the study of the synthesis of macromolecules and its regulation in eukaryotic cells.     

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.