Mutational Analyses of Translation Initiation Factor eIF1A in Saccharomyces cerevisiae

Saccharomyces cerevisiae에서 번역 개시 인자 eIF1A 돌연변이에 대한 분석

  • Kwon, Sung-Hun (Department of Biological Sciences, College of Natural Science, Inha University) ;
  • Kim, Jun-Ho (Department of Biological Sciences, College of Natural Science, Inha University) ;
  • Choi, Bo-Kyung (Department of Biological Sciences, College of Natural Science, Inha University) ;
  • Kim, Na-Yeon (Department of Biological Sciences, College of Natural Science, Inha University) ;
  • Choi, Do-Hee (Department of Biological Sciences, College of Natural Science, Inha University) ;
  • Park, Kyoung-Jun (Department of Biological Sciences, College of Natural Science, Inha University) ;
  • Eoh, Jung-Hyun (Department of Biological Sciences, College of Natural Science, Inha University) ;
  • Bae, Sung-Ho (Department of Biological Sciences, College of Natural Science, Inha University)
  • 권성훈 (인하대학교 자연과학대학 생명과학과) ;
  • 김준호 (인하대학교 자연과학대학 생명과학과) ;
  • 최보경 (인하대학교 자연과학대학 생명과학과) ;
  • 김나연 (인하대학교 자연과학대학 생명과학과) ;
  • 최도희 (인하대학교 자연과학대학 생명과학과) ;
  • 박경준 (인하대학교 자연과학대학 생명과학과) ;
  • 어정현 (인하대학교 자연과학대학 생명과학과) ;
  • 배성호 (인하대학교 자연과학대학 생명과학과)
  • Received : 2009.08.26
  • Accepted : 2009.09.21
  • Published : 2009.09.30

Abstract

Translation initiation factor eIF1A performs essential functions in various initiation steps including 43S preinitiation complex formation in eukaryotes, and contains a highly conserved oligonucleotide-binding (OB) fold. In our previous study, we discovered that eIF1A possesses RNA annealing activity and forms a stable complex with double-stranded RNA. In this study, we initiated site-directed mutations in eIF1A to find the active sites for these biochemical activities and to investigate whether they are essential functions for yeast cell growth. A truncated protein, eIF1A($\Delta$T), devoid of both N- and C-terminal domains but containing an intact OB-fold exhibited RNA annealing activity. In contrast, all point mutations in OB-fold domain, except R57D, impaired both RNA annealing and dsRNA binding activities, indicating that the intact OB-fold domain is required for both activities. Viabilities of the mutant yeast cells were not correlated with RNA annealing activity but with the in vivo protein stabilities in the case of R57D and K94D.

Acknowledgement

Supported by : 한국학술진흥재단

References

  1. Acker, M.G., B.S. Shin, J.S. Nanda, A.K. Saini, T.E. Dever, and J.R. Lorsch. 2009. Kinetic analysis of late steps of eukaryotic translation initiation. J. Mol. Biol. 385, 491-506 https://doi.org/10.1016/j.jmb.2008.10.029
  2. Battiste, J.L., T.V. Pestova, C.U.T. Hellen, and G. Wagner. 2000. The elF1A solution structure reveals a large RNA-binding surface important for scanning function. Mol. Cell 5, 109-119 https://doi.org/10.1016/S1097-2765(00)80407-4
  3. Baudin, A., O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, and C. Cullin. 1993. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 14, 3329-3330 https://doi.org/10.1093/nar/21.14.3329
  4. Cristofari, G. and J.L. Darlix. 2002. The ubiquitous nature of RNA chaperone proteins. Prog. Nucleic Acids Res. Mol. Biol. 72, 223-268 https://doi.org/10.1016/S0079-6603(02)72071-0
  5. Croitoru, V., K. Semrad, S. prenninger, L. Rajkowisch, M. Vejen, B.S. Laursen, H.U. Sperling-Petersen, and L.A. Isaksson. 2006. RNA chaperone activity of translation initiation factor IF1. Biochimie 88, 1875-1882 https://doi.org/10.1016/j.biochi.2006.06.017
  6. Fekete, C.A., D.J. Applefield, S.A. Blakely, N. Shirokikh, T. Pestova, J.R. Lorsch, and A.G. Hinnebusch. 2005. The eIF1A Cterminal domain promotes initiation complex assembly, scanning and AUG selection in vivo. EMBO J. 19, 3588-3601 https://doi.org/10.1038/sj.emboj.7600821
  7. Fekete, C.A., S.F. Mitchell, V.A. Cherkasova, D. Applefield, M.A. Algire, D. Maag, A.K. Saini, J.R. Lorsch, and A.G. Hinnebusch. 2007. N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection. EMBO J. 26, 1602-1614 https://doi.org/10.1038/sj.emboj.7601613
  8. Kainuma, M. and J.W.B. Hershey. 2001. Depletion and deletion analyses of eukaryotic translation initiation factor 1A in Saccharomyces cerevisiae. Biochimie 83, 505-514 https://doi.org/10.1016/S0300-9084(01)01279-2
  9. Kapp, L.D. and J.R. Lorsch. 2004. The molecular mechanics of eukaryotic translation. Annu. Rev. Biochem. 73, 657-658 https://doi.org/10.1146/annurev.biochem.73.030403.080419
  10. Kwon, S.H., I.H. Lee, N.Y. Kim, D.H. Choi, Y.M. Oh, and S.H. Bae. 2007. Translation initiation factor eIF1A possesses an RNA annealing activity in its oligonucleotide-binding fold. Biochem. Biophys. Res. Commun. 361, 681-686 https://doi.org/10.1016/j.bbrc.2007.07.084
  11. Li, W. and D.W. Hoffman. 2001. Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy. Protein Sci. 10, 2426-2438 https://doi.org/10.1110/ps.18201
  12. Lorsch, J.R. 2002. RNA chaperones exist and DEAD box proteins get a life. Cell 109, 797-800 https://doi.org/10.1016/S0092-8674(02)00804-8
  13. Maag, D., M.A. Algire, and J.R. Lorsch. 2006. Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection. J. Mol. Biol. 356, 724-737 https://doi.org/10.1016/j.jmb.2005.11.083
  14. Marintchev, A., V.G. Kolupaeva, T.V. Pestova, and G. Wagner. 2003. Mapping the binding interface between human eukaryotic initiation factors 1A and 5B: A new interaction between old partners. Proc. Natl. Acad. Sci. USA 100, 1535-1540 https://doi.org/10.1073/pnas.0437845100
  15. Mayer, O., L. Rajkowitsch, C. Lorenz, R. Konrat, and R. Schroeder. 2007. RNA chaperone activity and RNA-binding properties of the E. coli protein SptA. Nucleic Acids Res. 35, 1257-1269 https://doi.org/10.1093/nar/gkl1143
  16. Olsen, D.S., E.M. Savner, A. Mathew, F. Zhang, T. Krishnamoorthy, L. Phan, and A.G. Hinnebusch. 2003. Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo. EMBO J. 22, 193-204 https://doi.org/10.1093/emboj/cdg030
  17. Sette, M.P. van Tilborg, R. Spurio, R. Kaptein, M. Pici, C.O. Gualerzi, and R. Boelens. 1997. The structure of the translational initiation factor IF1 from E. coli contains an oligomer-binding motif. EMBO J. 16, 1436-1443 https://doi.org/10.1093/emboj/16.6.1436
  18. Sittka, A., V. Pfeiffer, K. Tedin, and J. Vogel. 2007. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium. Mol. Microbiol. 63, 193-217 https://doi.org/10.1111/j.1365-2958.2006.05489.x
  19. Tompa, P. and P. Csermely. 2004. The role of structural disorder in the function of RNA and protein chaperones. FASEB J. 18, 1169-1175 https://doi.org/10.1096/fj.04-1584rev
  20. Yu, Y., A. Marintchev, V.G. Kolupaeva, A. Unbehaun, T. Veryasova, S.C. Lai, P. Hong, G. Wagner, C.U.T. Hellen, and T.V. Pestova. 2009. Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing. Nucleic Acids Res. advance access published on June 26, 2009 https://doi.org/10.1093/nar/gkp519
  21. Zuniga, S., I. Sola, J.L. Moreno, P. Sabella, J. Plana-Duran, and L. Enjuanes. 2007. Coronavirus nucleocapsid protein is an RNA chaperone. Virology 357, 215-227 https://doi.org/10.1016/j.virol.2006.07.046