Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Primer RNA Synthesis by E. coli RNA Polymerase on the SSB-coated 229-nt ssi Signal of Lactococcal Plasmid pGKV21

Lactococcal plasmid pGKV21의 SSB-coated 229-nt ssi signal 상에서 E. coli RNA polymerase에 의한 시발체 RNA 합성

  • Jeong, Jin-Yong (Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Kim, Eun-Sil (Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Kim, Sam-Woong (Division of Biological Sciences, Pusan National University) ;
  • Kang, Ho-Young (Division of Biological Sciences, Pusan National University) ;
  • Bahk, Jeong-Dong (Division of Applied Life Sciences, Graduate School of Gyeongsang National University)
  • 정진용 (울산대학교 의과대학 서울아산병원 아산생명과학연구소) ;
  • 김은실 (울산대학교 의과대학 서울아산병원 아산생명과학연구소) ;
  • 김삼웅 (부산대학교 생명과학부) ;
  • 강호영 (부산대학교 생명과학부) ;
  • 박정동 (경상대학교 대학원 응용생명과학부)
  • Published : 2009.03.31
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Abstract

Plasmid pGKV21 contains a 229-nucleotide (nt) single-strand DNA initiation (ssi) signal. Using asymmetric PCR, we prepared a small single-stranded (ss) DNA fragment of the ssi signal and, using the 229-nt ssDNA fragment, determined the requirements of RNA polymerase for priming and DNA-protein interaction. The ssi fragment prepared was able to generate primer RNAs with almost the same efficiency as the $M13{\Delta}lac182/229$ phage DNA. However, the cssi (complementary strand of the ssi signal) fragment could not synthesize primer RNAs. This result suggests that the 229-nt ssi signal functions in a strand specific manner. Gel retardation and DNase I footprinting demonstrated that the synthesized ssi fragment could interact with both E. coli RNA polymerase and SSB protein to synthesize primer RNA. In Escherichia coli [pWVAp], an addition of rifampicin resulted in an accumulation of ssDNA, indicating that the host-encoded RNA polymerase is involved in the conversion of ssDNA to double-stranded plasmid DNA.

플라스미드 pGKV21에는 229-nt single-strand DNA initiation (ssi) signal이 존재한다. Asymmetric PCR 기법으로 합성된 229-nt ssDNA 단편을 이용하여 실제로 RNA polymerase에 의한 priming ability와 protein interaction을 확인하였다. in vitro primer RNA 합성 실험 결과, 229-nt ssDNA 단편은 filamentous M13 phage의 주형 DNA에서와 비슷한 효율로 시발체 RNA를 합성하였으며, 이 반응은 strand-specific하게 이루어졌다. DNase I footprinting과 gel retardation 실험 결과, RNA polymerase와 SSB 단백질은 229-nt ssDNA 단편에 stable interaction을 하며, 시발체 RNA를 합성하였다. 또한, in vivo 조건 하에서 RNA polymerase의 저해제인 rifampicin을 처리하여 세포내에 ssDNA 중간체가 집적되는 정도를 비교하여 본 결과, 플라스미드 pGKV21은 rifampicin-sensitive RNA polymerase가 상보가닥 합성에 관여 함을 보여 주었다.

Keywords

References

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