研究成果報告書

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2 20 6 UAA UGA UAG 20 Pyl. Pyl (AlocLys) N(epsilon)-benzyloxycarbonyl-lysine (ZLys) N(epsilon)-(o-azidobenzyloxycarbonyl) -lysine oazzlys (Yanagisawa et al., 2008b) N(epsilon)-(p-trifluoromethyldiazirinylbenzyloxy carbonyl)-lysine (TmdZLys) Yanagisawa et al., Mol. Biosyst. 8,3,202 A Pyl PylRS UAG trna Pyl mrna UAG Pyl (Blight et al., Nature 43, 333, 2004; Polycarpo et al., Proc.Nat.Acad.Sci. USA 0, 2450, 2004) PylRS Pyl trna Yanagisawa et al., Acta Crystallogr. F62, 03, 2006; Yanagisawa et al., J.Mol.Biol. 378, 634, 2008a PylRS Pyl PylRS polyspecific Yanagisawa et al., 2008a; Yanagisawa et al., Chem.Biol. 5, 87, 2008b trna (aars) trna aars aars trna trna (TyrRS)tRNA Tyr (CUA) Wang and Schultz, Ann.Rev.Biophys.Biomol.Struct. 35, 225, 2006; Liu and Schultz, Annu. Rev. Biochem. 79,43,200 PylRS trna Pyl aars trna Mukai et al., BBRC 37, 88, 2008; Hancock et al., J.Am.Chem.Soc.32, 489, 200; Chin, Annu. Rev. Biochem. 83,379, Fekner and Chan, Curr.Opin.Chem. Biol. 5, 387, 20; Wan and Liu, Biochim. Biophys.Acta.844, 059, 204 N(epsilon)-acetyl-lysine (AcLys)(Mukai et al., 2008) N(epsilon)-allyloxycarbonyl-lysine B. PylRS trna Pyl AB UAG Release factor (RF) trna(cua) UAG ORF 3 4 UAG RF RF UAG Neumann et al., Nature 464, 44, 200; Mukai et al., NAR 38, 888, 200; Wang et al., Nat.Chem.Biol. 20; Lajoie et al., Science 342, 357, 203 SSBC RF RFzero Mukai et al., Nucleic. Acids Res. 38,888,200; Mukai et al., BBRC, 4, 757, 20 Blank Mukai et al., NAR, 5, 9699, 205

3 X N(epsilon)- N A B C PylRS trna Pyl H3 H4 Mukai et al., 2008; Neumann et al., Mol.Cell 36, 53, 2009; Nguyen et al., JACS 3, 494, 2009; Wang et al., RFzero H4 K5 K8 K2 K6 mg H4 Mukai et al., 20 4 RFzero AlocLys AzZLys N(epsilon)-propargyloxy carbonyl-lysine (PocLys) RF. RFzero H4 RF RF PylRS trna Pyl RFzero RFzero H3 K4, K9, K27, K36, K79 4 AAA AAG UAG H3 BW253-RFzero UAG H3 PylRS(Y384F)tRNA Pyl ptk285-h3(amber)- PylRS(Y384F)-tRNA Pyl K2 BW253-RFzero 5 mm Boc N(epsilon)- methyl-n(epsilon)-t-butyloxycarbonyl-lysine [BocKme]37 OD 600 = % 42 H3-K4 K9 K27 K36 K79 BocKme H3(BocKme) 4 50%(TFA) BocKme Boc H3(Kme) 4 UAG pcr2.-h3(amber)bockme PylRS(Y384F) PylRS(Y309A/C348V/ Y384F) trna Pyl H3(BocKme) 2 H3 TFA Boc H3 H3

4 図５.複数リジンメチル化ヒストン H3 の調製スキーム４研究成果大腸菌 BW253-RFzero 株の発現系と PylRS trnapyl ペアを組み合わせることにより BocKme をヒストン H3 内の 4 カ所 K4 K9 K27 K36 K79 K4/27 K9/27 K4/36 K9/36 K4/27/36 K9/27/36 K4/27/36/79 K9/27/36/79 に導入し L 培地あたり 4 42 mg の H3(BocKme) 4 を得た図６図７. 脱 Boc 反応後のヒストン H3(Kme) 4 の質量分析図６. RFzero 株発現系を用いたメチルリジン誘導体 BocKme のヒストン H3 の複数カ所への導入とウエスタンブロット解析その後 TFA で H3 タンパク質上の Boc 基を脱保護することにより特定の位置に 4 個のモノメチルリジンが導入されたヒストン H3 を調製することに成功した H3 タンパク質内の BocKme およびモノメチルリジンは質量分析により確認した図 7 また BL2(DE3)-RFzero 株の粗抽出液を用いた無細胞タンパク質合成系により同様の方法で H3 内の 2 カ所 K4 K9 K27 K36 K4/27 にモノメチルリジンが導入された H3(BocKme) 2 を調製した図８ TFA 処理して脱 Boc したのち ESI-MS ウエスタンブロットでも正しい位置にモノメチルリジンが導入されていることが確認された図 9 また硫黄含有メチルリジン誘導体 Simon et al., Cell 28, 003, 2007 を含む H3 よりも本物のモノメチルリジン含有 H3 タンパク質の方が抗メチルリジン抗体の反応性は良いことが明らかとなった図 0 図８. RFzero 株の無細胞タンパク質合成系と PylRS 変異体 trnapyl の系によるヒストン H3-K4 K9 K27 K36 K4/K27 の位置への BocKme の導入青矢印は精製された H3 図９.無細胞タンパク質合成系で調製したヒストン H3 の TFA 処理前後の質量分析

5 0. H3 H3 Yanagisawa et al., 204a; Yanagisawa et al., 204b 3 Multiple site-specific installations of N(epsilon)-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis Yanagisawa, T., Takahashi, M., Mukai, T., Sato, S., Wakamori, M., Shirouzu, M., Sakamoto, K., Umehara, T., Yokoyama, S. ChemBioChem 5, (204). doi: 0.002/cbic Expanded genetic code technologies for incorporating modified lysine at multiple sites Yanagisawa, T., Umehara, T., Sakamoto, K., Yokoyama, S. ChemBioChem 5, (204). doi: 0.002/cbic A novel crystal form of pyrrolysyl-trna synthetase reveals the pre- and post-aminoacyl-trna synthesis conformational states of the adenylate and aminoacyl moieties and an asparagine residue in the catalytic site. Yanagisawa, T., Sumida, T., Ishii, R., Yokoyama, S. Acta Crystallogr. D69, 5-5 (203). doi: 0.07/S ) RF,2,3,2,2,2,2,2,2,4,2 2 CLST ( ) 2),2,3,4,3,2 2 3 CLST ) RF,2,3,2,2,2,2,2,2,4,2 2 CLST ) Expanded genetic code technologies for incorporating lysine residues with naturallyoccurring post-translational modifications at multiple sites in proteins. Tatsuo Yanagisawa Yoshifumi Fujii Kensaku Sakamoto 2 Takashi Umehara 2,3 Shigeyuki Yokoyama RIKEN Struct. Biol. Lab. 2 RIKEN CLST 3 JST, PRESTO RIKEN Epigenetics in Kobe ( CDB )) 5) RF,2,3,3,3,3,3,3,3,4,2 2 3 CLST ) Expanded Genetic Code Technologies for Incorporating Modified Lysine at Multiple Sites: Multiple Site-Specific Installations of N(epsilon) -Monomethyl-L-Lysine into Histone Proteins by Cell-Based and Cell-Free Protein Synthesis.

6 Tatsuo Yanagisawa,2, Mihoko Takahashi,3, Takahito Mukai,3, Shin Sato,3, Masatoshi Wakamori,3, Mikako Shirouzu,3, Kensaku Sakamoto,3, Takashi Umehara,3,4, Shigeyuki Yokoyama,2. ( RIKEN SSBC, 2 RIKEN Struct. Biol. Lab, 3 RIKEN CLST, 4 JST PRESTO,) trna Conference 204 ( Kyllini, Greece) 7) Synthesis of histone proteins with multiple and site-specific monomethyl-lysines Takashi Umehara,2,3, Tatsuo Yanagisawa 2,4, Mihoko Takahashi,2, Takahito Mukai,2, Shin Sato,2, Masatoshi Wakamori,2, Mikako Shirouzu,2, Kensaku Sakamoto,2, and Shigeyuki Yokoyama 2,4 ( RIKEN CLST, 2 RIKEN SSBC, 3 JST PRESTO, 4 RIKEN Struct. Biol. Lab.) FASEB meeting (204): Biological Methylation: Regulation of Chromatin, Epigenetics, and Disease Nassau, Bahamas 8) RF,2,3,3,3,3,3,3,3,4,2 SSBC 2 3 CLST 4 6 RNA ( ) 9),2,3,3,3,3,3,3,3,4,2 SSBC 2 3 CLST 4 9 ( ) 0) trna -trna Pyl Tatsuo Yanagisawa,3, Mihoko Takahashi 2,3, Shin Sato 2,3, Takahito Mukai 2,3, Masatoshi Wakamori 2,3, Mikako Shirouzu 2,3, Takashi Umehara 2,3, Kensaku Sakamoto 2,3, and Shigeyuki Yokoyama,2,3 RIKEN Struct. Biol. Lab., 2 RIKEN CLST, 3 RIKEN SSBC RIKEN EPIGENETICS in Yokohama ( ) ) Synthesis of histone with multiple site-specific monomethylation for reconstitution of epigenetic nucleosome Mihoko Takahashi,2, Tatsuo Yanagisawa 2,3, Shin Sato,2, Masatoshi Wakamori,2, Takahito Mukai,2, Mikako Shirouzu,2, Kensaku Sakamoto,2, Shigeyuki Yokoyama 2,3, Takashi Umehara,2,4 ( RIKEN CLST, 2 RIKEN SSBC, 3 JST PRESTO, 4 RIKEN Struct. Biol. Lab.) The 65th Fujihara Seminar; International Symposium on Synthetic Biology of Unnatural Base Pairs and Amino Acids (203 0 ) 2) trna trna,2 ( 2 ) 35 (BMB202) (202 2 ) YANAGISAWA TATSUO

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表1.eps Vol.1 C ontents 1 2 cell-free EMBO J Proc. Natl. Acad. Sci. USA NatureEMBO J 3 RNA NatureEMBO J Nature EMBO J 4 5 HCV HCV HCV HCV RNA HCV in situ 6 7 8 Nat Struct Mol Biol J Biol Chem Nat Commun J Virol