Dataset Information

Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y.

ABSTRACT: Histone H3.Y is conserved among primates. We previously reported that exogenously produced H3.Y accumulates around transcription start sites, suggesting that it may play a role in transcription regulation. The H3.Y nucleosome forms a relaxed chromatin conformation with flexible DNA ends. The H3.Y-specific Lys42 residue is partly responsible for enhancing the flexibility of the nucleosomal DNA. To our surprise, we found that H3.Y stably associates with chromatin and nucleosomes in vivo and in vitro. However, the H3.Y residues responsible for its stable nucleosome incorporation have not been identified yet. In the present study, we performed comprehensive mutational analyses of H3.Y, and determined that the H3.Y C-terminal region including amino acid residues 124-135 is responsible for its stable association with DNA. Among the H3.Y C-terminal residues, the H3.Y Met124 residue significantly contributed to the stable DNA association with the H3.Y-H4 tetramer. The H3.Y M124I mutation substantially reduced the H3.Y-H4 association in the nucleosome. In contrast, the H3.Y K42R mutation affected the nucleosome stability less, although it contributes to the flexible DNA ends of the nucleosome. Therefore, these H3.Y-specific residues, Lys42 and Met124, play different and specific roles in nucleosomal DNA relaxation and stable nucleosome formation, respectively, in chromatin.

SUBMITTER: Kujirai T

PROVIDER: S-EPMC5499910 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y.

Kujirai Tomoya T Horikoshi Naoki N Xie Yan Y Taguchi Hiroyuki H Kurumizaka Hitoshi H

Nucleus (Austin, Tex.) 20170124 3

Histone H3.Y is conserved among primates. We previously reported that exogenously produced H3.Y accumulates around transcription start sites, suggesting that it may play a role in transcription regulation. The H3.Y nucleosome forms a relaxed chromatin conformation with flexible DNA ends. The H3.Y-specific Lys42 residue is partly responsible for enhancing the flexibility of the nucleosomal DNA. To our surprise, we found that H3.Y stably associates with chromatin and nucleosomes in vivo and in vit ...[more]

PMID: 28118111

Similar Datasets

Project description:The nitrilase from Pseudomonas fluorescens EBC191 converted (R,S)-mandelonitrile with a low enantioselectivity to (R)-mandelic acid and (S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from Alcaligenes faecalis ATCC 8750 almost exclusively to (R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from P. fluorescens EBC191. The chimeric enzyme clearly resembled the nitrilase from A. faecalis ATCC 8750 in its turnover characteristics for (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile (2-PPN) and demonstrated an even higher enantioselectivity for the formation of (R)-mandelic acid than the nitrilase from A. faecalis. An alanine residue (Ala165) in direct proximity to the catalytically active cysteine residue was replaced in the nitrilase from P. fluorescens by a tryptophan residue (as found in the nitrilase from A. faecalis ATCC 8750 and most other bacterial nitrilases) and several other amino acid residues. Those enzyme variants that possessed a larger substituent in position 165 (tryptophan, phenylalanine, tyrosine, or histidine) converted racemic mandelonitrile and 2-PPN to increased amounts of the R enantiomers of the corresponding acids. The enzyme variant Ala165His showed a significantly increased relative activity for mandelonitrile (compared to 2-PPN), and the opposite was found for the enzyme variants carrying aromatic residues in the relevant position. The mutant forms carrying an aromatic substituent in position 165 generally formed significantly reduced amounts of mandeloamide from mandelonitrile. The important effect of the corresponding amino acid residue on the reaction specificity and enantiospecificity of arylacetonitrilases was confirmed by the construction of a Trp164Ala variant of the nitrilase from A. faecalis ATCC 8750. This point mutation converted the highly R-specific nitrilase into an enzyme that converted (R,S)-mandelonitrile preferentially to (S)-mandeloamide.

Project description:Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril's morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4'-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.

Dataset Information

Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y.

Publications

Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets