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Genetic encoding of isobutyryl-, isovaleryl-, and β-hydroxybutryl-lysine in E. coli.

ABSTRACT: Here we report the synthesis and genetic encoding of the lysine post translational modifications, β-hydroxybutyryl-lysine, isobutyryl-lysine and isovaleryl-lysine. The ability to obtain a homogenous protein samples with site-specific incorporation of these acylated lysine residues can serve as a powerful tool to study the biological role of lysine post translational modifications.

SUBMITTER: Christopher JA

PROVIDER: S-EPMC9706372 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Genetic encoding of isobutyryl-, isovaleryl-, and β-hydroxybutryl-lysine in <i>E. coli</i>.

Christopher Jayani A JA Galbada Liyanage Sahan A SA Nicholson Eve M EM Kinney William D WD Cropp T Ashton TA

RSC advances 20221129 53

Here we report the synthesis and genetic encoding of the lysine post translational modifications, β-hydroxybutyryl-lysine, isobutyryl-lysine and isovaleryl-lysine. The ability to obtain a homogenous protein samples with site-specific incorporation of these acylated lysine residues can serve as a powerful tool to study the biological role of lysine post translational modifications. ...[more]

PMID: 36545614

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[Dual genetic encoding of acetyl-lysine and non-deacetylatablethioacetyl-lysine mediated by flexizyme].

Project description: Not available

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Dual Genetic Encoding of Acetyl-lysine and Non-deacetylatable Thioacetyl-lysine Mediated by Flexizyme.

Project description:Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins in vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin type 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications.

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Genetically encoding thioacetyl-lysine as a non-deacetylatable analog of lysine acetylation in <i>Escherichia coli</i>.

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Genetic encoding of the post-translational modification 2-hydroxyisobutyryl-lysine.

Project description:We report the synthesis and genetic encoding of a recently discovered post-translational modification, 2-hydroxyisobutyryl-lysine, to the genetic code of E. coli. The production of homogeneous proteins containing this amino acid will facilitate the study of modification in full-length proteins.

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Unexpected implications of STAT3 acetylation revealed by genetic encoding of acetyl-lysine.

Project description:The signal transducer and activator of transcription 3 (STAT3) protein is activated by phosphorylation of a specific tyrosine residue (Tyr705) in response to various extracellular signals. STAT3 activity was also found to be regulated by acetylation of Lys685. However, the molecular mechanism by which Lys685 acetylation affects the transcriptional activity of STAT3 remains elusive. By genetically encoding the co-translational incorporation of acetyl-lysine into position Lys685 and co-expression of STAT3 with the Elk receptor tyrosine kinase, we were able to characterize site-specifically acetylated, and simultaneously acetylated and phosphorylated STAT3. We measured the effect of acetylation on the crystal structure, and DNA binding affinity and specificity of Tyr705-phosphorylated and non-phosphorylated STAT3. In addition, we monitored the deacetylation of acetylated Lys685 by reconstituting the mammalian enzymatic deacetylation reaction in live bacteria. Surprisingly, we found that acetylation, per se, had no effect on the crystal structure, and DNA binding affinity or specificity of STAT3, implying that the previously observed acetylation-dependent transcriptional activity of STAT3 involves an additional cellular component. In addition, we discovered that Tyr705-phosphorylation protects Lys685 from deacetylation in bacteria, providing a new possible explanation for the observed correlation between STAT3 activity and Lys685 acetylation.

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Ribosomes slide on lysine-encoding homopolymeric A stretches.

Project description:Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome 'sliding' represents an unexpected type of ribosome movement possible during translation.

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Translational control by lysine-encoding A-rich sequences.

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Multidrug-resistant E. coli encoding high genetic diversity in carbohydrate metabolism genes displace commensal E. coli from the intestinal tract.

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