Dataset Information

Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli.

ABSTRACT: Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl-tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic-code-expansion strategy to site-specifically incorporate N? -acetyl-l-lysine into selected positions of TyrRS for in vitro characterization. Enzyme assays demonstrated that acetylation at K85, K235, and K238 could impair the enzyme activity. In vitro deacetylation experiments showed that most acetylated lysine residues in TyrRS were sensitive to the E.?coli deacetylase CobB but not YcgC. In vitro acetylation assays indicated that 25 members of the Gcn5-related N-acetyltransferase family in E.?coli, including YfiQ, could not acetylate TyrRS efficiently, whereas TyrRS could be acetylated chemically by acetyl-CoA or acetyl-phosphate (AcP) only. Our in vitro characterization experiments indicated that lysine acetylation could be a possible mechanism for modulating aaRS enzyme activities, thus affecting translation.

SUBMITTER: Venkat S

PROVIDER: S-EPMC5629106 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli.

Venkat Sumana S Gregory Caroline C Gan Qinglei Q Fan Chenguang C

Chembiochem : a European journal of chemical biology 20170815 19

Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl-tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic-code-expansion strategy to site-specifically incorporate N<sup>ϵ</sup> -acetyl-l-lysine into selected positions of TyrRS for ...[more]

PMID: 28741290

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Project description:Nε-lysine acetylation is a common posttranslational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription factors, little is known about how this posttranslational modification regulates protein activity. In this work, we investigated how lysine acetylation affects translation in Escherichia coli. In multiple species of bacteria, ribosomal proteins are highly acetylated at conserved lysine residues, suggesting that this modification may regulate translation. In support of this hypothesis, we found that the addition of either of the acetyl donors acetyl phosphate and acetyl-coenzyme A inhibits translation but not transcription using an E. coli cell-free system. Further investigations using in vivo assays revealed that acetylation does not appear to alter the rate of translation elongation but, rather, increases the proportions of dissociated 30S and 50S ribosomes, based on polysome profiles of mutants or growth conditions known to promote lysine acetylation. Furthermore, ribosomal proteins are more acetylated in the disassociated 30S and 50S ribosomal subunits than in the fully assembled 70S complex. The effect of acetylation is also growth rate dependent, with disassociation of the subunits being most pronounced during late-exponential and early-stationary-phase growth-the same growth phase where protein acetylation is greatest. Collectively, our data demonstrate that lysine acetylation inhibits translation, most likely by interfering with subunit association. These results have also uncovered a new mechanism for coupling translation to the metabolic state of the cell. IMPORTANCE Numerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from central metabolite acetyl-coenzyme A or acetyl phosphate to a lysine residue in a protein. This posttranslational modification is known to regulate some central metabolic enzymes and transcription factors in bacteria, though a comprehensive understanding of its effect on cellular physiology is still lacking. In the present study, lysine acetylation was also found to inhibit translation in Escherichia coli by impeding ribosome association, most likely by disrupting salt bridges along the binding interface of the 30S and 50S ribosomal subunits. These results further our understanding of lysine acetylation by uncovering protein synthesis as a new target of regulation and aid in the design of bacteria for biotechnology applications where the growth conditions are known to promote lysine acetylation.

Dataset Information

Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli.

Publications

Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli.

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