title: Multiple site-specific installations of Nε-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis.
abstract: Lysine methylation is one of the important post-translational modifications of histones, and produces an N(ε) -mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N(ε)-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N(ε)-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl-protected N(ε)-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNA(Pyl)) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N(ε)-monomethyllysine residues. We installed N(ε)-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N(ε)-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N(ε)-monomethyllysine residue at position 27 better than the N(ε)-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N(ε)-monomethyllysines at multiple positions within a protein for large-scale production.
authors: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan).
authors: Tatsuo
authors: T
authors: Yanagisawa
authors: Mihoko
authors: M
authors: Takahashi
authors: Takahito
authors: T
authors: Mukai
authors: Shin
authors: S
authors: Sato
authors: Masatoshi
authors: M
authors: Wakamori
authors: Mikako
authors: M
authors: Shirouzu
authors: Kensaku
authors: K
authors: Sakamoto
authors: Takashi
authors: T
authors: Umehara
authors: Shigeyuki
authors: S
authors: Yokoyama
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism*; D005815:Genetic Code / Q000235:genetics; D006657:Histones / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D014176:Protein Biosynthesis
chemical_list: D000596:Amino Acids; D006657:Histones; D008239:Lysine
keywords: RNA; genetic code expansion; post-translational modifications; pyrrolysyl-tRNA synthetases; unnatural amino acids