Enlarging the Amino Acid Set of
            <i>Escherichia coli</i>
            by Infiltration of the Valine Coding Pathway

Döring, Volker; Mootz, Henning D.; Nangle, Leslie A.; Hendrickson, Tamara L.; Crécy‐Lagard, Valérie de; Schimmel, Paul; Marlière, Philippe

doi:10.1126/science.1057718

Cited by 187 publications

(181 citation statements)

References 28 publications

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“…The third codon, specifying Ser in the wild-type folA gene was changed to AAT (denoted folA:3 AAT S3N ) that is decoded by tRNA Asn . This is a permissive site for mutations in this protein since the presence of amino acid residues other than the wild-type Ser at position 3 does not affect the structure and function of the mature DHFR protein (41). Thus, in the absence of misacylated aa-tRNA, expression of the folA:3 AAT S3N allele will produce DHFR with a Ser3Asn substitution at position 3.…”

Section: Effective Missense Suppression Relies On Induction Of the Hementioning

confidence: 99%

Quality control despite mistranslation caused by an ambiguous genetic code

Ruan

Palioura

Sabina

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

131

132

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A high level of accuracy during protein synthesis is considered essential for life. Aminoacyl-tRNA synthetases (aaRSs) translate the genetic code by ensuring the correct pairing of amino acids with their cognate tRNAs. Because some aaRSs also produce misacylated aminoacyl-tRNA (aa-tRNA) in vivo, we addressed the question of protein quality within the context of missense suppression by Cys-tRNA Pro , Ser-tRNA Thr , Glu-tRNA Gln , and Asp-tRNA Asn . Suppression of an active-site missense mutation leads to a mixture of inactive mutant protein (from translation with correctly acylated aa-tRNA) and active enzyme indistinguishable from the wild-type protein (from translation with misacylated aa-tRNA). Here, we provide genetic and biochemical evidence that under selective pressure, Escherichia coli not only tolerates the presence of misacylated aa-tRNA, but can even require it for growth. Furthermore, by using mass spectrometry of a reporter protein not subject to selection, we show that E. coli can survive the ambiguous genetic code imposed by misacylated aa-tRNA tolerating up to 10% of mismade protein. The editing function of aaRSs to hydrolyze misacylated aa-tRNA is not essential for survival, and the EF-Tu barrier against misacylated aa-tRNA is not absolute. Rather, E. coli copes with mistranslation by triggering the heat shock response that stimulates nonoptimized polypeptides to achieve a native conformation or to be degraded. In this way, E. coli ensures the presence of sufficient functional protein albeit at a considerable energetic cost.accuracy ͉ aminoacyl-tRNA ͉ fidelity ͉ protein synthesis ͉ missense suppression

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Section: Effective Missense Suppression Relies On Induction Of the Hementioning

confidence: 99%

Quality control despite mistranslation caused by an ambiguous genetic code

Ruan

Palioura

Sabina

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

131

132

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“…Another strategy involves mutant synthetases with an enlarged substrate-binding site to accommodate those analogues that would not otherwise fit to the active site (Ibba and Hennecke, 1995). A third strategy is to modify the hydrolytic editing activity of the aminoacyl-tRNA synthetases so that the misaminocylation is not recognized as an error (Doring et al, 2001). An interesting modification of the misaminoacylation system is not to replace a single amino acid with an analogue, but to re-assign only a single codon to code for an analogue and thus break the degeneracy of the genetic code (Kwon et al, 2003).…”

Section: Residue-specific Incorporation Of Non-canonical Amino Acids mentioning

confidence: 99%

Gelatinase-mediated migration and invasion of cancer cells

Björklund

Koivunen

2005

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer

465

609

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“…The hs mt LeuRS appears to lack editing activity and achieves aminoacylation fidelity solely using its precise synthetic active site, which is not characteristic of other bacterial and eukaryotic LeuRSs studied to date (6)(7)(8)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)38). The hs mt LeuRS must possess special features within its active site that provide the uniquely high levels of amino acid specificity.…”

Section: Roles Of a Flexible Domain Containing A Critical Residue In mentioning

confidence: 99%

“…the loss of this editing activity, with translational inaccuracies leading to perturbed cellular function (6)(7)(8).…”

mentioning

confidence: 99%

A Single Residue in Leucyl-tRNA Synthetase Affecting Amino Acid Specificity and tRNA Aminoacylation

Lue

2007

Biochemistry

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Human mitochondrial leucyl-tRNA synthetase (hs mt LeuRS) achieves high aminoacylation fidelity without a functional editing active site, representing a rare example of a class I aminoacyl-tRNA synthetase (aaRS) that does not proofread its products. Previous studies demonstrated that the enzyme achieves high selectivity by using a more specific synthetic active site that is not prone to errors under physiological conditions. Interestingly, the synthetic active site of hs mt LeuRS displays a high degree of homology with prokaryotic, lower eukaryotic and other mitochondrial LeuRSs that are less specific. However, there is one residue that differs between hs mt and Escherichia coli (E. coli) LeuRSs located on a flexible closing loop near the signature KMSKS motif. Here we describe studies indicating that this particular residue (K600 in hs mt LeuRS and L570 in E. coli LeuRS) strongly impacts aminoacylation in two ways -it affects both amino acid discrimination and transfer RNA (tRNA) binding. While this residue may not be in direct contact with the amino acid or tRNA substrate, substitutions of this position in both enzymes leads to altered catalytic efficiency and perturbations to the discrimination of leucine and isoleucine. In addition, tRNA recognition and aminoacylation is affected. These findings indicate that the conformation of the synthetic active site -modulated by this residue -may be coupled to specificity and provide new insights into the origins of selectivity without editing.Aminoacyl-tRNA synthetases (aaRSs) 1 are important translational factors. They catalyze the covalent attachment of amino acids to their cognate tRNAs, an essential step in the translation of the genetic code (1-3). The fidelity of protein synthesis is dependent upon the accuracy with which aaRSs discriminate between cognate and noncognate amino acids and numerous cellular tRNAs.To explain the high fidelity for cognate amino acids exhibited by aaRSs, a "double sieve" model has been proposed. This model, relevant mainly to class I aaRSs, relies on the use of two functionally independent active sites to achieve amino acid selectivity (4,5). In the first synthetic active site, amino acids are recognized, activated with ATP, converted to aminoacyl adenylates, and then transferred to tRNA. Amino acids larger than the cognate substrate are excluded from this site by sterics; smaller amino acids present a more significant problem as they can be bound, misactivated, and used erroneously to acylate tRNA. To resolve these errors, some aaRSs utilize a second editing active site that proofreads the products made by the activation site and hydrolytically cleaves substrates containing non-cognate amino acids. Both pre-transfer editing of misactivated aminoacyl adenylates and post-transfer editing of misacylated tRNA can occur at this second active site. Many systems are severely affected by *To whom correspondence should be addressed: Phone: 617-552-3121, Fax: 617-552-2705, E-mail: shana.kelley@bc.edu. Many previous studies have focused ...

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Enlarging the Amino Acid Set of Escherichia coli by Infiltration of the Valine Coding Pathway

Cited by 187 publications

References 28 publications

Quality control despite mistranslation caused by an ambiguous genetic code

Quality control despite mistranslation caused by an ambiguous genetic code

Gelatinase-mediated migration and invasion of cancer cells

A Single Residue in Leucyl-tRNA Synthetase Affecting Amino Acid Specificity and tRNA Aminoacylation

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