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Journal of Virology, September 2006, p. 9181-9191, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.00295-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Pseudoknot in a Preactive Form of a Viral RNA Is Part of a Structural Switch Activating Minus-Strand Synthesis

Department of Cell Biology and Molecular Genetics, University of Maryland—College Park, College Park, Maryland 20742,¹ Center for Cancer Research Nanobiology Program, National Cancer Institute, Frederick, Maryland 21702²

Received 9 February 2006/ Accepted 30 June 2006

RNA can adopt different conformations in response to changes in the metabolic status of cells, which can regulate processes such as transcription, translation, and RNA cleavage. We previously proposed that an RNA conformational switch in an untranslated satellite RNA (satC) of Turnip crinkle virus (TCV) regulates initiation of minus-strand synthesis (G. Zhang, J. Zhang, A. T. George, T. Baumstark, and A. E. Simon, RNA 12:147-162, 2006). This model was based on the lack of phylogenetically inferred hairpins or a known pseudoknot in the "preactive" structure assumed by satC transcripts in vitro. We now provide evidence that a second pseudoknot (₂), whose disruption reduces satC accumulation in vivo and enhances transcription by the TCV RNA-dependent RNA polymerase in vitro, stabilizes the preactive satC structure. Alteration of either ₂ partner caused nearly identical structural changes, including single-stranded-specific cleavages in the pseudoknot sequences and strong cleavages in a distal element previously proposed to mediate the conformational switch. These results indicate that the preactive structure identified in vitro has biological relevance in vivo and support a requirement for this alternative structure and a conformational switch in high-level accumulation of satC in vivo.

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