Identification of the +1 ribosomal frameshifting site of LRV1-4 by mutational analysis

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 al., 1996). However, the exact frameshift site has not been identified. In this study, we compared the frameshift efficiency of a 259bp (n...

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Bibliographic Details
Published in:Archives of pharmacal research 2005, 28(8), , pp.956-962
Main Authors: Kim, Se Na, Choi, Jung Ho, Park, Min Woo, Jeong, Sun Joo, Han, Kyung Sook, Kim, Hong Jin
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Binding Sites
DNA Mutational Analysis
Frameshifting, Ribosomal
Genes, Reporter
Leishmaniavirus - genetics
Luciferases
Molecular Sequence Data
Nucleic Acid Conformation
Open Reading Frames
Plasmids
RNA, Double-Stranded - genetics
RNA, Viral - genetics
Software
Viral Proteins - genetics
약학
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Summary: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 al., 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 71bp (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 71bp 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 71bp region where ORF2 and ORF3 overlap, and that nt. 2644-2659 (the upward hairpin stem) play a key role in generating the +1 frameshift.
ISSN:0253-6269
1976-3786
DOI:10.1007/BF02973883