Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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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
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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.

번역 개시 인자 eIF1A는 진핵생물에서 43S preinitiation complex 형성을 비롯한 번역 개시 과정의 여러 단계에서 필수적인 역할을 하며, 잘 보존된 oligonucleotide-binding (OB) fold를 가지고 있는 단백질이다. 본 연구진은 이전 연구에서 eIF1A가 RNA annealing 활성을 가지고 있으며 double-stranded RNA에 결합하여 안정된 복합체를 형성한다는 것을 발견한 바 있다. 본 연구에서는 이러한 활성을 나타내는데 필요한 active site를 찾고, 이러한 활성이 효모의 성장에 필수적인 기능인지를 알아보기 위하여 여러 가지 돌연변이를 제조하였다. N-말단과 C-말단은 제거되었지만 완전한 OB-fold를 가지고 있는 eIF1A($\Delta$T)는 RNA annealing 활성을 보이는 반면, OB-fold에 돌연변이가 도입된 단백질들은 모두 활성이 사라졌다. 또한, R57D 돌연변이를 제외한 모든 OB-fold 돌연변이는 dsRNA에도 결합하지 않았다. 이러한 결과는 eIF1A의 RNA annealing 활성과 dsRNA 결합에는 완전한 OB-fold domain이 필요하다는 것을 의미한다. 돌연변이들이 효모의 성장에 미치는 영향을 조사한 결과, RNA annealing 활성과 효모의 성장은 뚜렷한 연관성이 없었으며, 적어도 R57D와 K94D 경우에는 돌연변이가 성장하지 못하는 원인이 생체 내 eIF1A 단백질의 안정성과 관계있는 것으로 생각된다.

Keywords

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