Creation of a Yeast Strain with Co‐Translationally Acylated Nucleosomes

Abstract

Structurally diverse acylations have been identified as post-translational modifications (PTMs) on histone lysine residues, but their functions and regulations remain largely unknown. Interestingly, in nature, a lysine acylation analog, pyrrolysine, is introduced as a co-translational modification (CTM) through genetic encoding. To explore this alternative life form, we created a model organism Saccharomyces cerevisiae containing site-specific lysine CTMs (acetyllysine, crotonyl-lysine, or another synthetic analog) at histone H3K56 using non-canonical amino acid mutagenesis to afford a chemically modified nucleosome in lieu of their own in vivo. We further demonstrated that acetylation of histone H3K56 partly tends to provide a more favorable chromatin environment for DNA repair in yeast compared to crotonylation and crosstalk with other PTMs differently. This study provides a potentially universal approach to decipher the consequences of different histone lysine PTMs in eukaryotes.

Co‐translational Installation of Posttranslational Modifications by Non‐canonical Amino Acid Mutagenesis

“…Incorporation of AcK into H3.3 protein resulted in gene transcription level changes that are likely regulated by acetylation. A similar study was carried out in yeast cells using plasmid‐supplied genetic components to compare the effects of acetylation and crotylation at K56 in the H3 protein [144] . The encoded AcK provided better protection against genotoxic reagents and led to the upregulation of genes mainly functioning in cell wall and sexual reproduction pathways when treated with methyl methanesulfonate.…”

“…A similar study was carried out in yeast cells using plasmid-supplied genetic components to compare the effects of acetylation and crotylation at K56 in the H3 protein. [144] The encoded AcK provided better protection against genotoxic reagents and led to the upregulation of genes mainly functioning in cell wall and sexual reproduction pathways when treated with methyl methanesulfonate. The acetylation at K56 also changes the transcription level of PTM enzymes.…”

Studying Reversible Protein Post‐translational Modification through Co‐translational Modification

“…The Chin group firstly engineered Mb PylRS/tRNA CUA pair to generate homogenously acetylated proteins and enable the first lysine PTM (acetyl‐lysine, AcK) incorporated into proteins produced in E. coli [31] . Following this pioneer work, chemical biologists have been devoted to evolving GCE systems derived from archean PylRS/tRNA Pyl and Ec LeuRS/tRNA Leu pair so that they can efficiently recognize and encode other acylated lysine into histones, for example, formyl‐lysine (ForK), [32] propionyl‐lysine (PrK), [33] crotonyl‐lysine (CroK), [33b,34] butyryl‐lysine (BuK), [33] 2‐hydroxyisobutyrylation (HibK) [35] and benzoylation (BzK), [36] and achieve genetic encoding of AcK in yeast, [34b,37] mammalian cells, [38] and even animals [39] . However, the negatively charged state of succinyl group makes it particularly difficult to evolve an amino‐acyl tRNA synthetases (aaRS) that can recognize succinyl‐lysine (SuK) in living cells.…”

“…Several non‐hydrolyzable acetyllysine analogs have been genetically incorporated into desired positions of proteins in E. coli and yeast by using engineered PylRS variants, such as 2‐amino‐8‐oxononanoic acid (KetoK), [49] thio‐acetyllysine (TAcK) [50] and trifluoro‐acetyllysine (TFAcK) [51] . The easily prepared S ‐(4‐oxopentyl)‐ l ‐cysteine (KetoTK) might have the same resistance to deacetylases as KetoK and can be encoded by Ec LeuRS variant in yeast [34b] and mammalian cells [52] (Figure 2). Furthermore, deacetylase inhibitors (nicotinamine) [31] can also be added to growth media to prevent deacetylation.…”

Genetically Encoded Epitope Tag for Probing Lysine Acylation-Mediated Protein–Protein Interactions

“…Investigation of a given PTM can be challenging as it may require homogeneous substrate proteins decorated by modification(s) of interest site specifically. Incorporation of unnatural amino acids (UAAs) through amber codon-mediated genetic code expansion (GCE) offers a powerful strategy to tackle the challenge. − Although the incorporation of Kac and Kcr through GCE has been achievable and should indeed be helpful to interrogate the YEATS–Kac/Kcr interactions, , their applications may be limited by the following reasons. The PPIs mediated by Kac/Kcr with YEATS domains are normally weak (with dissociation constants in the micromolar to submillimolar levels), and the Kac/Kcr marks installed by GCE are not resistant to removal catalyzed by endogenous deacylases, making the PPIs even dynamic and transient.…”