scholarly journals The effect of trimethoprim on macromolecular synthesis in Escherichia coli. General effects on ribonucleic acid and protein synthesis

1973 ◽  
Vol 136 (2) ◽  
pp. 225-234 ◽  
Author(s):  
R. J. Smith ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Rna Synthesis ◽  
Protein Chain ◽  
Macromolecular Synthesis ◽  
Chain Initiation ◽  
Chain Polymerization ◽  
Rna Accumulation ◽  
New Protein ◽  
Endogenous Synthesis

In trimethoprim-inhibited RCstr strains of Escherichia coli, the expression of the RC control of stable RNA synthesis arose primarily from a decrease in the intracellular concentrations of glycine and methionine, and not from inhibition of the initiation of new protein chains. In non-supplemented cultures, experiments with rifampicin showed that the immediate response to the addition of trimethoprim was a rapid decrease in the rate of initiation of RNA chains. This was followed after a few minutes by a sufficiently large fall in the rate of endogenous synthesis of nucleotide bases to cause a decrease in the rate of RNA chain polymerization. Inhibition of RNA chain initiation was thus overridden by an accumulation of DNA-dependent RNA polymerases upon the cistrons. RCrel strains also accumulated polymerases upon the DNA in similar circumstances, but did not suffer the initial effects on chain initiation. If purines were supplied before adding trimethoprim, RCstr strains polymerized RNA chains at normal rates, but initiation rates were permanently decreased. In either situation, an increased% of the RNA formed was mRNA. However, in RCrel strains supplemented with bases, trimethoprim did not affect either the rate of initiation of new chains or their rates of polymerization or the relative rates of synthesis of stable RNA and mRNA. Protein synthesis was also severely inhibited by trimethoprim. Though the addition of glycine and methionine to base-supplemented, trimethoprim-inhibited RCstr strains did not apparently affect the decreased rate of protein synthesis, RNA accumulation resumed at its normal rate. Thus, the inhibition of protein chain initiation had no effect on the rate of RNA accumulation in either RCstr or RCrel bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.

scholarly journals The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Regulation of ribonucleic acid synthesis by ‘magic spot’ nucleotides

1973 ◽  
Vol 136 (2) ◽  
pp. 249-257 ◽  
Author(s):  
R. J. Smith ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Rna Synthesis ◽  
Control Mechanism ◽  
Acid Synthesis ◽  
Macromolecular Synthesis ◽  
Guanosine Tetraphosphate ◽  
Additional Control ◽  
High Concentrations ◽  
Rna Accumulation ◽  
Rate Of Accumulation

During the inhibition of RCstr, but not RCrel mutants of Escherichia coli by trimethoprim the unusual nucleotides MSI (guanosine tetraphosphate, ppGpp) and MSII rapidly accumulated. The production of these nucleotides was not dependent on the addition of nucleotide base supplements to RCstr cultures before trimethoprim, and the MSI nucleotide concentrations in non-supplemented or purine-supplemented cultures were comparable with the concentrations obtained when the cells were inhibited with l-valine (1g/l). Rifampicin rapidly decreased MSI and MSII nucleotide concentrations in trimethoprim-inhibited cultures to the basal values. Several situations were noted, in which MS nucleotide concentrations in trimethoprim-inhibited RCstr cells could be drastically lowered without giving rise to an immediate resumption of stable RNA accumulation. If RCstr mutants were first inhibited with trimethoprim and then given purines 15min later, MS nucleotide concentrations fell rapidly, because of a temporarily enhanced rate of accumulation of stable RNA. However, after a further 5min, RNA accumulation stopped, though MS nucleotide concentrations remained low. Also, if either glycine or methionine were added to trimethoprim-inhibited cultures supplemented with purines, RNA accumulation did not resume, though MS nucleotide concentrations rapidly declined. With both amino acids present, there was both a decline in MS nucleotide concentration and a resumption in stable RNA synthesis. These findings suggest that MSI nucleotide concentrations in trimethoprim-inhibited bacteria are not the sole factors in the control of stable RNA synthesis. It is possible that, during the period when the RCstr cells contained high concentrations of MS nucleotides, some factor important in the MSI-mediated control of stable RNA synthesis was irreversibly inactivated. However, as antibiotics (e.g. chloramphenicol) both abolished high MS nucleotide concentrations and permitted a rapid resumption of stable RNA accumulation in the same conditions, it is more likely that an additional control mechanism has come into play.

scholarly journals The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Ribosome maturation in RCstr and RCrel strains

1973 ◽  
Vol 136 (2) ◽  
pp. 235-247 ◽  
Author(s):  
J. E. M. Midgley ◽  
R. J. Smith
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
High Rate ◽  
Chain Elongation ◽  
Chain Initiation ◽  
Rna Accumulation ◽  
Ribosomal Protein Synthesis ◽  
Nucleotide Bases

When Escherichia coli was inhibited with trimethoprim in media supplemented with nucleotide bases, glycine and methionine, both RCstr and RCrel strains continued to accumulate RNA at rates very close to those in growing controls. The effects of trimethoprim on protein synthesis were studied by using as an experimental basis the rate of maturation of ribosomal particles from RNA-rich precursors. 1. In RCstr cultures given nucleotide bases but no amino acids, RNA accumulation was inhibited because of amino acid lack. However, maturation of ribosomes from their precursors was more severely inhibited than was the synthesis of rRNA. The restraints on protein synthesis were more severe at the level of translation than the transcription of operons specific for the formation of ribosomal proteins. The kinetic delay time in the passage of rRNA from RNA-rich intermediates to the final ribosome products was therefore increased some three- to four-fold. 2. In RCrel cultures in the same conditions, trimethoprim inhibition stopped ribosomal particle synthesis, but rRNA-rich precursors accumulated. 3. If glycine+methionine were also added to inhibited RCstr cultures, RNA accumulation resumed at a high rate. However, ribosomal maturation was still considerably disturbed because of a disproportionate response of the cells in the formation of protein and RNA. 4. With RCrel cultures, addition of the amino acids caused a large increase in the rate of ribosome maturation, though the degree of disproportionation between the rates of rRNA and ribosomal protein synthesis was now identical with that found in RCstr strains. 5. When inhibited RCrel cultures were supplemented, there was still a severe inhibition of protein synthesis at the level of chain initiation, but inaccuracies in the process of polypeptide chain elongation were greatly decreased. This suggests that the effects of the RCrel mutation on the fidelity of protein synthesis in bacteria are not directed at the point of chain initiation.

Hypoxanthine-xanthine oxidase-related defect in polypeptide chain initiation by endothelium

1989 ◽  
Vol 66 (1) ◽  
pp. 450-457 ◽  
Author(s):  
L. Jornot ◽  
A. F. Junod
Keyword(s):  
Protein Synthesis ◽  
Xanthine Oxidase ◽  
Rna Synthesis ◽  
Polypeptide Chain ◽  
Total Proteins ◽  
Initiation Complex ◽  
Chain Initiation ◽  
Oxidase System ◽  
Initiation Factors ◽  
Related Defect

After exposure to O2 intermediates generated by the hypoxanthine-xanthine oxidase system (HX-XO), the rate of [3H]phenylalanine incorporation into total proteins in cultured endothelial cells was markedly reduced. This reduction, which was prevented by catalase, could not be explained by 1) changes in amino acid pools, 2) increased rate of degradation of newly synthesized proteins, 3) impaired poly(A)+ RNA synthesis and efficiency, 4) decreased rate of amino acylation. On the other hand, the increase in the monoribosome-to-polyribosome ratio suggested that translation was affected at the level of chain initiation. Further analysis indicated that 40S initiation complex formation was normal, whereas the assembly of 80S initiation complex was inhibited. Results from reconstitution experiments showed that both normal and treated ribosomes could support normal protein synthesis in the presence of normal initiation factors (IFs). In contrast, IFs from HX-XO lysates did not support normal protein synthesis with ribosomes from either source. Thus, the effect of XO treatment on protein synthesis appears to be an initiation defect related to decreased IF activity and/or availability.

scholarly journals A New Protein Synthesis Factor from Escherichia coli

1970 ◽  
Vol 67 (3) ◽  
pp. 1137-1142 ◽  
Author(s):  
Y. W. Kan ◽  
F. Golini ◽  
R. E. Thach
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
New Protein

scholarly journals Precommitted erythroid cells enriched in cultures of suboptimally induced Friend erythroleukemia cells

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1565-1571
Author(s):  
E DiMambro ◽  
M Galanti ◽  
SB Levy
Keyword(s):  
Protein Synthesis ◽  
Rna Synthesis ◽  
Terminal Differentiation ◽  
Metabolic Inhibitors ◽  
Hemoglobin Synthesis ◽  
Erythroleukemia Cells ◽  
Chemically Induced ◽  
Primed Cell ◽  
Murine Erythroleukemia ◽  
New Protein

Abstract In the presence of suboptimal inducing amounts of dimethylsulfoxide or hexamethylenebisacetamide, a major proportion of uncommitted murine erythroleukemia (MEL) cells was found to be precommitted or primed for commitment, which was demonstrated by their rapid commitment to terminal differentiation when recultured for short periods of time (three to six hours) with fresh inducer. These same cells did not commit if left in the original inducer-containing media or if replated in fresh media without inducer. The two inducers could be interchanged in the priming and postpriming period without affecting the commitment event. However, hemin, an agent that induces hemoglobin synthesis without commitment, showed no ability to enhance commitment of a primed cell population. The rapid commitment of primed cells was inhibited by 12-O-tetradecanoylphorbol-13-acetate and cordycepin but not by cycloheximide. The latter finding indicated that this rapid inducer- dependent commitment event required new RNA synthesis but not new protein synthesis. The precommitment state was lost within six hours of the growth of cells in the absence of inducer but could be sustained longer if cells were incubated in cycloheximide. These studies characterize a precommitment state not previously described and one that appears during chemically induced differentiation in the absence of metabolic inhibitors. The stabilization of these precommitted cells by cycloheximide suggests that the reversibility of precommitment involves new protein synthesis. These findings show that MEL cells proceed to terminal differentiation by accumulating unstable products that must be maintained by the inducer until the final commitment event.

Insulin stimulates macromolecular synthesis in cultured glial cells from rat brain

1985 ◽  
Vol 249 (5) ◽  
pp. C484-C489 ◽  
Author(s):  
D. W. Clarke ◽  
F. T. Boyd ◽  
M. S. Kappy ◽  
M. K. Raizada
Keyword(s):  
Protein Synthesis ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Rat Brain ◽  
Glial Cells ◽  
Rna Synthesis ◽  
Macromolecular Synthesis ◽  
Total Response ◽  
Dose Dependent ◽  
Stimulation Of

The effect of insulin on macromolecular synthesis in glial cells cultured from brains of 1-day-old rats was studied to investigate the role of insulin in brain growth. Insulin caused a dose-dependent stimulation of protein synthesis (measured by [3H]valine incorporation into protein) that became significant by 7 nM insulin. Maximal stimulation of protein synthesis of 145% of control occurred with 18 nM insulin. Long-term protein synthesis was also stimulated to 136% of control by insulin in a dose-dependent manner after 6 days of insulin incubation. Insulin also stimulated net RNA and DNA synthesis (measured by [3H]uridine and [3H]thymidine incorporation into RNA or DNA, respectively) with significant stimulation by 2 nM insulin. Net RNA synthesis stimulation was maximal at 120% of control by 18 nM insulin. Plateau stimulation of DNA synthesis of 175% of control was reached by 200 nM insulin. The effects of insulin on glial protein and RNA synthesis appear to be mediated completely by the insulin receptor. Insulin, in physiological concentrations, stimulated glial DNA synthesis via its interaction with the insulin receptor (46% of total response). At supraphysiological concentrations insulin may have stimulated DNA synthesis via its cross-reactivity with the insulinlike growth factor I receptor (54% of total response). Thus insulin, at concentrations known to exist in the brain, stimulates the processes necessary for growth in the glial cell and is an important growth factor in the developing rat brain.

THE EFFECT OF ACTINOBOLIN ON NUCLEIC ACID AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI

1966 ◽  
Vol 12 (3) ◽  
pp. 515-520 ◽  
Author(s):  
D. E. Hunt ◽  
R. F. Pittillo ◽  
E. P. Johnson ◽  
F. C. Moncrief
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Nucleic Acid ◽  
Mechanism Of Action ◽  
Rna Synthesis ◽  
The Other ◽  
Cross Resistance ◽  
Inhibition Of Protein Synthesis

Actinobolin inhibits protein synthesis in Escherichia coli. When the antibiotic is added to a culture at the time of inoculation, RNA synthesis is also inhibited. Inhibition of RNA synthesis appears to be a consequence of inhibition of protein synthesis. Cross-resistance experiments suggest that the mechanism of action of actinobolin differs from that of the other inhibitors of protein synthesis, chloramphenicol and sparsomycin. Phenylalanine prevents the action of actinobolin provided the amino acid and antibiotic are added simultaneously; this effect is not observed if the phenylalanine is added 1 hour after the addition of the antibiotic. Evidence is presented that the mechanism by which phenylalanine prevents inhibition by actinobolin differs from that which has been suggested for azaserine and p-fluorophenylalanine.

scholarly journals Precommitted erythroid cells enriched in cultures of suboptimally induced Friend erythroleukemia cells

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1565-1571
Author(s):  
E DiMambro ◽  
M Galanti ◽  
SB Levy
Keyword(s):  
Protein Synthesis ◽  
Rna Synthesis ◽  
Terminal Differentiation ◽  
Metabolic Inhibitors ◽  
Hemoglobin Synthesis ◽  
Erythroleukemia Cells ◽  
Chemically Induced ◽  
Primed Cell ◽  
Murine Erythroleukemia ◽  
New Protein

In the presence of suboptimal inducing amounts of dimethylsulfoxide or hexamethylenebisacetamide, a major proportion of uncommitted murine erythroleukemia (MEL) cells was found to be precommitted or primed for commitment, which was demonstrated by their rapid commitment to terminal differentiation when recultured for short periods of time (three to six hours) with fresh inducer. These same cells did not commit if left in the original inducer-containing media or if replated in fresh media without inducer. The two inducers could be interchanged in the priming and postpriming period without affecting the commitment event. However, hemin, an agent that induces hemoglobin synthesis without commitment, showed no ability to enhance commitment of a primed cell population. The rapid commitment of primed cells was inhibited by 12-O-tetradecanoylphorbol-13-acetate and cordycepin but not by cycloheximide. The latter finding indicated that this rapid inducer- dependent commitment event required new RNA synthesis but not new protein synthesis. The precommitment state was lost within six hours of the growth of cells in the absence of inducer but could be sustained longer if cells were incubated in cycloheximide. These studies characterize a precommitment state not previously described and one that appears during chemically induced differentiation in the absence of metabolic inhibitors. The stabilization of these precommitted cells by cycloheximide suggests that the reversibility of precommitment involves new protein synthesis. These findings show that MEL cells proceed to terminal differentiation by accumulating unstable products that must be maintained by the inducer until the final commitment event.

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