Amino Acid Pool of Escherichia coli during the Different Phases of Growth.

Raunio, Raimo; Rosenqvist, Heikki

doi:10.3891/acta.chem.scand.24-2737

Cited by 52 publications

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“…The valine concentration in the E. coli cells is about fivetimes higher than the isoleucine concentration [17,181. The discrimination at these concentrations is also described in Fig.…”

Section: Resultsmentioning

confidence: 87%

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

Airas

1992

European Journal of Biochemistry

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A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E .possible. If the E . Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E . Ile-AMp with E . and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E . l'e-AMP and the corrected *E$&Mp. If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be benefitial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.The activation of amino acids by the aminoacyl-tRNA synthetases produces PP,, which is able to bind to the aminoacyl adenylate in the reversc reaction [I]. Both the activation and the pyrophosphorolysis are rapid reactions [2] which allow an equilibrium at this step in the presence of PPi. The PPi strongly shifts the equilibrium from the aminoacyl adenylate intermediates towards the start of the total reaction. PP, is decomposed by the inorganic pyrophosphatase present in all living cells. The pyrophosphatase, however, does not destroy all PP,, but a moderate concentration is maintained. The PP, concentration in Escherichia roli cells, thoroughly studied in this laboratory [3 -51, is about 0.5 mM and is not changed very much by varying the conditions.The amino acid specificity of the aminoacyl-tRNA synthetases has been described to be increased in four steps [6]. Two initial discrimination steps occur before the activation reaction, the pretransfer proofreading works at the aminoacyl adenylate step and the posttransfer proofreading at the aminoacyl-tRNA step (Eqn 1).The effect of PP, on the aminoacyl-tRNA synthetase reaction has already been considered in two earlier papers on this work [7, 81. The present work uses the kinetic equations derived in the preceding paper [9]. The roles of PP, in the initial discriminations and pretransfer proofreading were calculated. MATERIALS AND METHODS EquationsThe rate equations were derived as suggested by Cha [lo] and as described in detail in the preceding paper [9]. The reaction scheme was divided in separate rapid equilibrium segments. Since the amino acid specificity is to be examined, the binding reaction for the amino acid makes one boundary of the segments, and the different amino acids, isoleucine and valine, have their own segments (Eqn 2).

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“…The valine concentration in the E. coli cells is about fivetimes higher than the isoleucine concentration [17,181. The discrimination at these concentrations is also described in Fig.…”

Section: Resultsmentioning

confidence: 87%

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

Airas

1992

European Journal of Biochemistry

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“…Novel co-inducer mediated regulation of gcvTHP centration of glycine is approximately 41 µM (Raunio & Rosenqvist, 1970). This level is apparently insufficient to allow full de-repression of the gcv operon as seen in the gcvR mutant GS1053λgcvT ::lacZ, but is sufficient to bind some GcvR and to allow de-repression above the basal level seen in the gcvA mutant GS998λgcvT ::lacZ.…”

mentioning

confidence: 98%

Glycine binds the transcriptional accessory protein GcvR to disrupt a GcvA/GcvR interaction and allow GcvA-mediated activation of the Escherichia coli gcvTHP operon

Heil

Stauffer

2002

Microbiology

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The Escherichia coli gcvTHP operon is under control of the LysR-type transcriptional regulator GcvA. GcvA activates the operon in the presence of glycine and represses the operon in its absence. Repression by GcvA is dependent on a second regulatory protein, GcvR. Generally, LysR-type transcriptional regulators bind to specific small co-effector molecules which results in either their altered affinity for specific binding sites on the DNA or altered ability to bend the DNA, resulting in either activation or repression of their respective operons. This study shows that glycine, the co-activator for the gcv operon, does not alter either GcvA's ability to bind DNA nor its ability to bend DNA. Rather, glycine binds to GcvR, disrupting a GcvA/GcvR interaction required for repression and allowing GcvA activation of the gcvTHP operon. Amino acid changes in GcvR that reduce glycine binding result in a loss of glycine-mediated activation in vivo.

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“…The extraction was carried out at pH 10 and at 100 "C for 10 min as described by Raunio & Rosenqvist (1970). For the extraction, cells in 70 ml of suspension were centrifuged at 5000 g for 10 min in a cold room, and they were not washed.…”

Section: R P R a U N I O And M L E P P A V I R T A Methodsmentioning

confidence: 99%

“…Separation of free amino acids from the extract and from the medium was carried out on a Dowex 50 W (Hf-form) column, as described by Raunio & Rosenqvist (1970). Amino acids were analysed as heptafluorobutyryl amino acid propyl esters by gas-liquid chromatography.…”

Section: R P R a U N I O And M L E P P A V I R T A Methodsmentioning

confidence: 99%

“…The changes in the endogenously-synthesized amino acid pools of bacteria have been dealt with in several papers (Raunio & Rosenqvist, 1970;Lowry, Carter, Ward & Glaser, 197 I ;Clark, Peterson & Barnlohr, 1972). Mandelstam (1958) showed that growth rate, energy metabolism and nitrogen starvation had an influence on the amino acid pool, and amino acids were found in the medium only in the case of nitrogen starvation.…”

Section: Introductionmentioning

confidence: 99%

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The Effect of Culture Age, Chloramphenicol and B6 Inhibitors on Intra- and Extracellular Keto and Amino Acids of Escherichia coli B

Raunio

Uvirta

1975

Journal of General Microbiology

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S U M M A R YKeto acids and free amino acids were assayed in the cells and the medium of Escherichia coli B growing in the presence of chloramphenicol, cycloserines, aminooxyacetate, and limiting nitrogen source.Under these growth-limiting conditions the cells accumulated ketoglutarate and 'ketovaline' but no other keto acids. In all experiments only ketoglutarate, pyruvate, and 'ketovaline' were found in the medium. Amino acids are released into the medium in the early phases of growth and the composition of the extracellular amino acids is similar to that of the amino acid pool. The concentrations of free amino acids were 103-104 times higher in the cell than in the medium. The internal pool composition is fixed under all growth-limiting conditions. In the presence of the drugs the cells release amino acids into the medium.

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Amino Acid Pool of Escherichia coli during the Different Phases of Growth.

Cited by 52 publications

References 0 publications

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

Glycine binds the transcriptional accessory protein GcvR to disrupt a GcvA/GcvR interaction and allow GcvA-mediated activation of the Escherichia coli gcvTHP operon

The Effect of Culture Age, Chloramphenicol and B6 Inhibitors on Intra- and Extracellular Keto and Amino Acids of Escherichia coli B

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