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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)
Publication Information
Archives of Pharmacal Research / v.28, no.8, 2005 , pp. 956-962 More about this Journal
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.
Keywords
LRV (Leishmania virus)1-4; Ribosomal +1 frameshift; Luciferase activity;
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