A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate

Richardson, Charles J.; First, Eric A.

doi:10.1016/j.ab.2015.05.008

Cited by 12 publications

(21 citation statements)

References 41 publications

(47 reference statements)

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“…The Fluorothreonyl-tRNA Selectivity of FthB Is Primarily Mediated by k cat . To further characterize the selectivity of FthB between fluorothreonyl-and threonyl-tRNA, a spectrophotometric assay employing a coupled aminoacylation/deacylation system was developed based on a similar assay reported for TyrRS (35). In this assay, the steadystate rate of deacylation was measured by coupling the hydrolysis of aminoacyl-tRNA to the aminoacylation reaction that occurs upon release of the free tRNA.…”

Section: Resultsmentioning

confidence: 99%

Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya

McMurry

Chang

2017

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

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Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small molecules, ranging from therapeutics to materials. In contrast, only a few fluorinated compounds made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered in While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochemical, genetic, physiological, and proteomic studies, we show that FthB is a-acting transfer RNA (tRNA) editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. While -acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metabolism. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.

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Section: Resultsmentioning

confidence: 99%

Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya

McMurry

Chang

2017

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

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“…We have demonstrated that the tyrosyl-tRNA synthetase assay can be used to monitor the aminoacylation of tRNA by either l - or d -tyrosine, with cyclodityrosine synthase and d -tyrosyl-tRNA deacylase being used to cleave the l -Tyr-tRNA and d -Tyr-tRNA products, respectively. A detailed description of this assay can be found in [1] .…”

Section: Data Experimental Design Materials and Methodsmentioning

confidence: 99%

“…This requirement for stoichiometric amounts of tRNA can be alleviated if the aminoacyl-tRNA product is cleaved following the tRNA aminoacylation reaction, regenerating the free tRNA substrate. This data article is related to the research article entitled “A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate” in which this approach is used to develop a continuous spectrophotometric assay for tyrosyl-tRNA synthetase [1] . Here we present enzymes that can be used to cleave the aminoacyl-tRNA product for at least 16 of the 20 naturally occurring amino acids.…”

mentioning

confidence: 99%

Expanding a tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases

Richardson

First

2015

Data in Brief

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Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. In general, aminoacyl-tRNA synthetase assays require stoichiometric amounts of tRNA, which limits their sensitivity while increasing their cost. This requirement for stoichiometric amounts of tRNA can be alleviated if the aminoacyl-tRNA product is cleaved following the tRNA aminoacylation reaction, regenerating the free tRNA substrate. This data article is related to the research article entitled “A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate” in which this approach is used to develop a continuous spectrophotometric assay for tyrosyl-tRNA synthetase [1]. Here we present enzymes that can be used to cleave the aminoacyl-tRNA product for at least 16 of the 20 naturally occurring amino acids. These enzymes can be used to extend the tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases.

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“…The sensitivity of these assays can be increased with aa-tRNA recycling systems, which utilize aa-tRNAs as substrates and regenerate the tRNAs needed for the aaRS reaction ( e.g. , tRNA-dependent pathways for synthesis of cyclodipeptide [20] and modified lipids [21], or aa-tRNA editing activities [22], see text for details). FFluc: firefly luciferase, luc: luciferin, AMPD: adenosine monophosphate deaminase, IMPDH: inosine monophosphate dehydrogenase, PPase: pyrophosphatase, PNPase: purine nucleoside phosphorylase, AMMP: 2-amino-6-mercapto-7-methyl purine, MESG: AMMP ribonucleoside, Ribose-1P: ribose 1 phosphate.…”

Section: Introductionmentioning

confidence: 99%

“…For instance, enzymes that recycle the tRNA for additional rounds of aminoacylation increase the amount of ATP consumed, or AMP and PPi produced (Figure 1). This strategy was used to increase ATP consumption in an assay for TyrRS, by adding cyclodityrosine synthase or D-tyrosyl-tRNA Tyr deacylase, which utilize Tyr-tRNA Tyr to synthesize a cyclodipeptide or to hydrolyze the aa-tRNA, respectively [20]. A similar strategy was developed for AlaRS using alanyl-diacylaglycerol synthase, which uses Ala-tRNA Ala as an aa donor for synthesis of alanyl-diacylglycerol [21].…”

Section: Introductionmentioning

confidence: 99%

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

Grube

Roy

2017

RNA Biology

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Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNAs to produce the aminoacyl-tRNAs (aa-tRNAs) required by ribosomes for translation of the genetic message into proteins. To ensure the accuracy of tRNA aminoacylation, and consequently the fidelity of protein synthesis, some aaRSs exhibit a proofreading (editing) site, distinct from the aa-tRNA synthetic site. The aaRS editing site hydrolyzes misacylated products formed when a non-cognate amino acid is used during tRNA charging. Because aaRSs play a central role in protein biosynthesis and cellular life, these proteins represent longstanding targets for therapeutic drug development to combat infectious diseases. Most existing aaRS inhibitors target the synthetic site, and it is only recently that drugs targeting the proofreading site have been considered. In the present study, we developed a robust assay for the high-throughput screening of libraries of inhibitors targeting both the synthetic and the proofreading sites of up to four aaRSs simultaneously. Thus, this assay allows for screening of eight distinct enzyme active sites in a single experiment. aaRSs from several prominent human pathogens (i.e., Mycobacterium tuberculosis, Plasmodium falciparum, and Escherichia coli) were used for development of this assay.

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A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate

Cited by 12 publications

References 41 publications

Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya

Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya

Expanding a tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

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