A radioactive assay for the physiological activity of the tryptophan synthetase α subunit in crude extracts of Escherichia coli

Sorensen, Penny; Hijazi, Kathryn A.; Murgola, Emanuel J.; Buckingham, Richard H.

doi:10.1016/0300-9084(90)90005-2

Cited by 8 publications

(4 citation statements)

References 18 publications

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“…IGP was freshly prepared as described [8]. Tryptophan synthetase was assayed in the IGP to Trp conversion with [ 3 H]serine (28.0 Ci/mmol) [15]. Enzymatic unit is defined as the amount producing 1 μmol of tryptophan/mg of protein.…”

Section: Methodsmentioning

confidence: 99%

In vivo formation of glutamyl‐tRNA^Gln in Escherichia coli by heterologous glutamyl‐tRNA synthetases

et al. 2003

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Two types of glutamyl-tRNA synthetase exist: the discriminating enzyme (D-GluRS) forms only Glu-tRNA Glu , while the non-discriminating one (ND-GluRS) also synthesizes Glu-tRNA Gln , a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). Testing the capacity to complement a thermosensitive E. coli gltX mutant and to suppress an E. coli trpA49 missense mutant we examined the properties of heterologous gltX genes. We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu-tRNA Glu , A. ferrooxidans and Helicobacter pylori GluRS2 form Glu-tRNAGln in E. coli in vivo.

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Section: Methodsmentioning

confidence: 99%

In vivo formation of glutamyl‐tRNA^Gln in Escherichia coli by heterologous glutamyl‐tRNA synthetases

et al. 2003

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“…IGP was freshly prepared as described [8]. Tryptophan synthetase was assayed in the IGP to Trp conversion with [ 3 H] serine (28.0 Ci/mmol) [17]. Enzymatic unit is defined as the amount producing 1 lmol of tryptophan/mg of protein, and expressed as percentage of mutant enzyme activity relative to that of the wild type TrpA in E. coli strain W3110.…”

Section: Suppression Of E Coli Ftp5822 Trpa Nonsense Mutationmentioning

confidence: 99%

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

et al. 2006

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In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA Pyl by pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA Pyl . We report here the in vitro aminoacylation of tRNA Pyl by PylRS with two Pyl analogues: -prolyl-lysine) and N-e-cyclopentyloxycarbonyl-L L-lysine (Cyc). Escherichia coli, transformed with the tRNA Pyl and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of D D-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA Pyl and D D-prolyl-lysine-tRNA Pyl as substrates during protein synthesis. Furthermore, the formation of active b-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.

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“…The cultures were grown at 37°C to late log phase, harvested by centrifugation, washed twice with ice-cold NaCl (0.9%) solution, and resuspended in buffer A (0.05 M KPO 4 [pH 7.0], 0.1 mg of pyridoxal-5-phosphate per ml, 10 mM 2-mercaptoethanol). Cell extracts were prepared (18), dialyzed against buffer A containing 50% glycerol, and stored at Ϫ20°C. IGP was freshly prepared as described by Mosteller (10 …”

Section: Plasmids and Strainsmentioning

confidence: 99%

Protein Synthesis in Escherichia coli with Mischarged tRNA

Min¹,

Kitabatake²,

Polycarpo³

et al. 2003

J Bacteriol

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Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA Asp , while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA Asn , a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA Asn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA Asn are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition (7). The discriminating enzyme (D-AspRS) recognizes only tRNA Asp , while the nondiscriminating one (ND-AspRS) also recognizes tRNA Asn and therefore forms both Asp-tRNA Asn and Asp-tRNA Asp . Most bacteria and archaea lack asparaginyl-tRNA synthetase and are unable to synthesize Asn-tRNA Asn by direct acylation of tRNA. These organisms rely on the ND-AspRS to produce the misacylated Asp-tRNA Asn , which is then converted by a tRNA-dependent amidotransferase to the correctly acylated Asn-tRNA Asn (1,4,5,19). Thus, the ND-AspRS is essential in organisms that form Asn-tRNA by transamidation.The primary sequence distinguishes two general groups of AspRS. There is a predominantly bacterial type of AspRS that is about 580 amino acids, in addition to a shorter archaealeukaryotic type of about 430 amino acids. In vitro data have made clear that discriminating and nondiscriminating enzymes exist in both groups (16,20). The determinants in the protein sequence responsible for tRNA discrimination are not known.The two AspRS types are usually separated in nature. Genome analyses of bacteria and archaea have revealed that the presence of the ND-AspRS is always accompanied by the occurrence of the heterotrimeric GatCAB amidotransferase, an enzyme capable of converting the misacylated Asp-tRNA Asn to Asn-tRNA Asn (2,5,19). Presumably, this is to avoid introducing the misacylated Asp-tRNA Asn into an organism's translational apparatus and potentially endangering protein synthesis. This reasoning is supported by the fact that the heterologous expression of ND-AspRS or ND-GluRS in Escherichia coli, which lacks GatCAB, is highly toxic to the cell, especially when the synthetase genes are overexpressed (15). However, some organisms (e.g., Deinococcus radiodurans and Thermus thermophilus) contain a D-AspRS in addition to an ND-AspRS and a GatCAB amidotransferase (1,3,5,9).We wanted to observe how E. coli copes with in vivo mischarging effected by the ND-AspRS, as this organism is unable to eliminate the toxic Asp-tRNA Asn . Therefore, we developed an approach that would, in fact, require E. coli to be dependent on the presence of mischarged Asp-tRNA Asn for growth. To this aim, we used missense suppression of a specific mutation in the trp...

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A radioactive assay for the physiological activity of the tryptophan synthetase α subunit in crude extracts of Escherichia coli

Cited by 8 publications

References 18 publications

In vivo formation of glutamyl‐tRNA^Gln in Escherichia coli by heterologous glutamyl‐tRNA synthetases

In vivo formation of glutamyl‐tRNA^Gln in Escherichia coli by heterologous glutamyl‐tRNA synthetases

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

Protein Synthesis in Escherichia coli with Mischarged tRNA

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A radioactive assay for the physiological activity of the tryptophan synthetase α subunit in crude extracts of Escherichia coli

Cited by 8 publications

References 18 publications

In vivo formation of glutamyl‐tRNAGln in Escherichia coli by heterologous glutamyl‐tRNA synthetases

In vivo formation of glutamyl‐tRNAGln in Escherichia coli by heterologous glutamyl‐tRNA synthetases

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

Protein Synthesis in Escherichia coli with Mischarged tRNA

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In vivo formation of glutamyl‐tRNA^Gln in Escherichia coli by heterologous glutamyl‐tRNA synthetases

In vivo formation of glutamyl‐tRNA^Gln in Escherichia coli by heterologous glutamyl‐tRNA synthetases