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1 pp.! mrna trna RNA tmrna mrna RNA trna mrna tmrna tmrna tmrna Ray Molecular mechanism of trans-translation Hyouta Himeno, Daisuke Kurita, Kazuma Takada, Takayuki Konno, Kyoko Hanawa-Suetsugu, Chie Takemoto, Masahito Kawazoe, Shigeyuki Yokoyama, Nobukazu Nameki, Gota Kawai, Akira Muto Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho, Hirosaki, Japan; RIKEN Genomic Sciences Center,Suehiro-cho, Tsurumi, Yokohama, Japan; Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryuu, Japan; Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, Chiba, Japan Apirion tmrna SsrA RNA Sa RNA tmrna trna tmrna a tmrna trna CCA T trna trna tmrna trna RNase P tmrna trna Ala G-U A a mrna trna PKPK

2 trna tmrna mrna Tu tmrna tmrna tmrna tmrna Keiler in vivo mrna C in vitro tmrna trna mrna mrna trna atmrna trna T b RNA RNA tmrna tmrna trna mrna tmrna trna trna Ala trna trna AlaRS tmrna tmrna trna tmrna Tu tmrna tmrna trna Ala mrna mrna tmrna mrna tmrna tmrna mrna tmrna trna mrna tmrna in vitro polyu polyu tmrna

3 in vitro tmrna polyu tmrna mrna polyphe C G-U A-U tmrna tmrna tmrna tmrna trna mrna mrna trna RNA b tmrna tmrna tmrna mrna tmrna tmrna tmrna Keiler C ClpAP ClpXP FtsH Tsp Tsp ATP SspB ClpXP ClpXP mrna mrna tmrna Neisseria DNA Caulobacter tmrna tmrna tmrna tmrna tmrna nonstop mrna decaynsd mrna no-go mrna decayngd mrna tmrna tmrna RNase P AlaRSEF-TuSSmpB RNase P tmrna AlaRS tmrna trna

4 tmrna SmpB SmpB tmrna tmrna SPrsA VacBRNase RYfbG RNase R mrna Caulobacter RNase R SmpB tmrna in vitro tmrna GTP EF-Tu trnaaa-trna T EF-Tuaa-tRNAGTP A EF-Tu aa-trna T Ala-tmRNA S SD mrna mrna mrna S tmrna PK PK SmpB kda NMR Aquifex aeolicus Thermus thermophilus SmpB asmpb tmrna SmpB polyupolyphe tmrna in vitro in vitro in vitro in vitro mrna trnaef-tuef-g Ala-tmRNA SmpB polyu UUC mrna Phe-tRNA Phe EF-TuEF-G mrna poly Phe Ala-tmRNA SmpB polyphe-ala-tmrna Ala-tRNA Ala polyphe-ala-ala-trna Ala A P E trna ATAAAPPPPEE mrna trna

5 anmr T. thermophilus SmpB PDB IDJH β β C NMR bx A. aeolicus SmpBtRNA PDB IDPV trnapdb IDTNAtRNA trna SmpB trna trna SmpB β β SmpB C

6 trna tmrna trna mrna mrnatrna tmrna trna A mrna tmrna tmrna trna mrna tmrna A tmrna tmrna Ala-tmRNA mrna A trna UGA mrna in vitro A mrna A -trnarfrrfala-tmrna in vivo mrna A Ala-tmRNA -trna RF A mrna tmrna A mrna tmrna RelE MazFChpAK A RNase RelE MazF A mrna RNase A mrna in vitro mrna tmrna SD AUG GCA A tmrna PKPKPK PK PK PK PK A SmpB tmrna SmpBA SmpB S S

7 Wower tmrna S mrna S GC S tmrna S tmrna S in vivo S SmRNA tmrna S S in vitro -trna Ala-tmRNA -Ala-tmRNA -trna S SmpB SmpB in vitro SmpB polyu SmpB SmpB tmrna SmpB in vitro tmrna in vivo tmrna tmrna trna SmpB SmpB SmpB NMR SmpBtRNA X b SmpB tmrna trna tmrna tmrna tmrna tmrna SmpB tmrna SmpB SmpB SmpB tmrna SmpB tmrna SmpB tmrna SmpB C tmrna C C NMR SmpB A P SmpB SmpB trna SmpBtRNA X SmpB trna CCA SmpB C mrna A P trna SmpBtRNAmRNA SmpB trna C mrna tmrna SmpB SD P mrna P trna P

8 SmpB trna C mrna SmpB A SmpB T. thermophilus tmrna SmpBEF-TuGDP SmpB SmpB A SmpB C SmpB SmpB GTPase EF-Tu SmpB tmrna SmpB trna mrna SmpB trnamrna mrna tmrna SmpBC trna tmrna SmpB mrna mrnasmpbtmrna tmrna trna Ray, B.K. & Apirion, D.Mol. Gen. Genet.,,. Ushida, C., Himeno, H., Watanabe, T., & Muto, A. Nucleic Acids Res.,,. Komine, Y., Kitabatake, M., Yokogawa, T., Nishikawa, K., & Inokuchi, H.Proc. Natl. Acad. Sci. USA,,. Felden, B., Himeno, H., Muto, A., McCutcheon, J., Atkins, J. F., & Gesteland, R.F.RNA,,. Felden, B., Hanawa, K., Atkins, J.F., Himeno, H., Muto, A., Gesteland, R.F., McClosky, J.A., & Crain, P.F.EMBO J.,,. Muto, A., Ushida, C., & Himeno, H.Trends Biochem. Sci.,,. Keiler, K.C., Shapiro, L., & Williams, K.P.Proc. Natl. Acad. Sci. USA,,. Tadaki, T., Fukushima, M., Ushida, C., Himeno, H., & Muto, A.FEBS Lett.,,. Tu, G.F., Reid, G.E., Zhang, J.G., Moritz, R.L., & Simpson, R. J.J. Biol. Chem.,,. Keiler, K.C., Waller, P.R.H., & Sauer, R.T.Science,,. Muto, A., Sato, M., Tadaki, T., Fukushima, M., Ushida, C., & Himeno, H.Biochimie,,. Himeno, H., Sato, M., Tadaki, T., Fukushima, M., Ushida, C., & Muto, A.J. Mol. Biol.,,. Nameki, N., Tadaki, T., Muto, A., & Himeno, H.J. Mol. Biol.,,. Gottesman, S., Roche, E., Zhou, Y., & Sauer, R.T. Genes Dev.,,. Herman, C., Thevenet, D., Bouloc, P., Walker, G.C., & DAri, R.Genes Dev.,,. Levchenko, I., Seidel, M., Sauer, R.T., & Baker, T.A. Science,,. Withey, J.H. & Friedman, D.I.Annu. Rev. Microbiol.,,. Yamamoto, Y., Sunohara, T., Jojima, K., Inada, T., & Aiba, H. RNA,,. Retallack, D.M. & Friedman, D.I.Cell,,.

9 Huang, C., Wolfgang, M.C., Withey, J., Koomey, M., & Friedman D.I.EMBO J.,,. Muto, A., Fujihara, A., Ito, K., Matsuno, J., Ushida C., & Himeno, H.Genes Cells,,. Withey,J.&Friedman,D.J.J. Bacteriol.,,. Ranquet, C., Geiselmann, J., & Toussaint, A.Proc. Natl. Acad. Sci. USA,,. Julio, S.M., Heithoff, D.M., & Mahan, M.J.J. Bacteriol.,,. Retallack, D.M. & Friedman, D.I.Cell,,. Keiler, K.C. & Shapiro, L.J. Bacteriol.,,. Fujihara, A., Tomatsu, H., Inagaki, S., Tadaki, T., Ushida, C., Himeno, H., & Muto, A.Genes Cells,,. Jacob, Y., Seif, E., Paquet, P.O., & Lang, B.F.RNA,,. Frischmeyer, P.A., van Hoof, A., ODonnell, K., Guerrerio, A. L., Parker, R., & Dietz, H.C.Science,,. Doma,M.K.&Parker,R.Nature,,. Barends,S.,Karzai,A.W.,Sauer,R.T.,Wower,J.,&Kraal,B. J. Mol. Biol.,,. Tedin, K., Resch, A., & Blasi, U.Mol. Microbiol.,,. Wower, J., Zwieb, C., Guven, S.A., & Wower, I. EMBO J.,,. Dong, G., Nowakowski, J., & Hoffman, D.W.EMBO J.,,. Someya, T., Nameki, N., Hosoi, H., Suzuki, S., Hatanaka, H., Fujii, M., Terada, T., Shirouzu, M., Inoue, Y., Shibata, T., Kuramitsu, S., Yokoyama, S., & Kawai, G. FEBS Lett.,,. Karzai, A.W., Susskind, M.M., & Sauer, R.T.EMBO J.,,. Karzai, A.W. & Sauer, R.T.Proc. Natl. Acad. Sci. USA,,. Richards, J., Mehta, P., & Karzai, A.W.Mol. Microbiol.,,. Hong, S.J., Tran, Q.A., & Keiler, K.C.Mol. Microbiol.,,. Shimizu, Y. & Ueda, T.The role of SmpB protein in trans-translation. FEBS Lett.,,. Ivanova, N., Pavlov, M.Y., Felden, B., & Ehrenberg, M. J. Mol. Biol.,,. Asano, K., Kurita, D., Takada, K., Konno, T., Muto, A., & Himeno, H.Nucleic Acids Res.,,. Roche, E.D. & Sauer, R.T.EMBO J.,,. Roche, E.D. & Sauer, R.T. J. Biol. Chem.,,. Hayes, C.S., Bose, B., & Sauer, R.T.Proc. Natl. Acad. Sci. USA,,. Hayes, C.S., Bose, B., & Sauer, R.T.J. Biol. Chem.,,. Collier, J., Binet, E., & Bouloc, P.Mol. Microbiol.,,. Pedersen, K., Zavialov, A.V., Pavlov, M.Y., Elf, J., Gerdes, K., & Ehrenberg, M.Cell,,. Christensen, S.K., Pedersen, K., Hansen, F.G., & Gerdes, K. J. Mol. Biol.,,. Nameki, N., Tadaki, T., Himeno, H., & Muto, A. FEBS Lett.,,. Nameki, N., Felden, B., Atkins, J.F., Gesteland, R.F., Himeno, H.,&Muto,A.J. Mol. Biol.,,. Nameki, N., Chattopadhyay, P., Himeno, H., Muto, A., & Kawai, G.Nucleic Acids Res.,,. Lee, S., Ishii, M., Tadaki, T., Muto, A., & Himeno, H. RNA,,. Takahashi, T., Konno, T., Muto, A., & Himeno, H.J. Biol. Chem.,,. Konno, T., Takahashi, T., Kurita, D., Muto, A., & Himeno, H. Nucleic Acids Res.,,. Bordeau, V. & Felden, B.Biochimie,,. Valle, M., Gillet, R., Kaur, S., Henne, A., Ramakrishnan, V., & Frank, J.Science,,. Ito, K., Tadaki, T., Lee, S., Takada, K., Muto, A., & Himeno, H.FEBS Lett.,,. McGinness, K.E. & Sauer, R.T.Proc. Natl. Acad. Sci. USA,,. Sengupta, J., Agrawal, R.K., & Frank, J.Proc. Natl. Acad. Sci. USA,,. Takada, K., Takemoto, C., Kawazoe, M., Konno, T., Hanawa- Suetsugu, K., Lee, S., Shirouzu, M., Yokoyama, S., Muto, A., & Himeno, H.RNA, in press. Hanawa-Suetsugu, K., Takagi, M., Inokuchi, H., Himeno, H., & Muto, A.Nucleic Acids Res.,,. Hanawa-Suetsugu, K., Bordeau, V., Himeno, H., Muto, A., & Felden, B.Nucleic Acids Res.,,. Nameki, N., Someya, T., Okano, S., Suemasa, R., Kimoto, M., Hanawa-Suetsugu, K., Terada, T., Shirouzu, M., Hirao, I., Takaku, H., Himeno, H., Muto, A., Kuramitsu, S., Yokoyama, S., & Kawai, G.J. Biochem.,,. Gutmann, S., Haebel, P.W., Metzinger, L., Sutter, M., Felden, B., & Ban, N.Nature,,. Wower, I.K., Zwieb, C., & Wower, J.J. Biol. Chem.,,. Hallier, M., Ivanova, N., Rametti, A., Pavlov, M., Ehrenberg, M., & Felden, B.J. Biol. Chem.,,. Sundermeier, T.R., Dulebohn, D.P., Cho, H.J., & Karzai, A.W. Proc. Natl. Acad. Sci. USA,,. Jacob, Y., Sharkady, S.M., Bhardwaj, K., Sanda, A.A., & Williams, K.P.J. Biol. Chem.,,. Kaur, S., Gillet, R., Li, W., Gursky, R., & Frank, J. Proc. Natl. Acad. Sci. USA,,.

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