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The Pharmaceutical Society of Korea (대한약학회)
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Identification of the+1 Ribosomal Frameshifting Site of LRV1-4 by Mutational Analysis

  • Kim Se Na (College of Pharmacy, Chung Ang University) ;
  • Choi Jung Ho (College of Pharmacy, Chung Ang University) ;
  • Park Min Woo (Department of Molecular Biology, Dankook University) ;
  • Jeong Sun Joo (Department of Molecular Biology, Dankook University) ;
  • Han Kyung Sook (School of Computer Science and Engineering, Inha University) ;
  • Kim Hong Jin (College of Pharmacy, Chung Ang University)
  • Published : 2005.08.01
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Abstract

Leishmania virus (LRV)1-4 has been reported to produce a fusion of ORF2 and ORF3 via a programmed +1 frameshift in the region where ORF2 and ORF3 overlap (Lee et a/., 1996). However, the exact frameshift site has not been identified. In this study, we compared the frameshift efficiency of a 259bp (nt. 2565-2823), frameshift region of LRV1-4, and the 71 bp (nt. 2605-2678) sub-region where ORF2 and ORF3 overlap. We then predicted the frameshift site using a new computer program (Pseudoviewer), and finally identified the specific region associated with the mechanism of the LRV1-4's+1 frameshift by means of a mutational analysis based on the predicted structure of LRV1-4 RNA. The predicted structure was confirmed by biochemical analysis. In order to measure the frameshift efficiency, constructs that generate luciferase without a frameshift or with a+1 frameshift, were generated and in vitro transcription/translation analysis was performed. Measurements of the luciferase activity generated, showed that the frameshift efficiency was about $1\%$ for both the 259bp (LRV1-4 259FS) and 71 bp region (LRV1-4 71FS). Luciferase activity was strongly reduced in a mutant (LRV1-4 NH: nt. 2635-2670) with the entire hairpin deleted and in a mutant (LRV1-4 NUS: nt. 2644-2659) with the upper stem of the hairpin deleted. These results indicate that the frameshift site in LRV1-4's is in the 71 bp region where ORF2 and ORF3 overlap, and that nt. 2644-2659 (the upward hairpin stem) playa key role in generating the +1 frameshift.

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References

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