| Cis-acting Elements in the 3` Region of Potato virus X are Required for HostProtein Binding |
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Sun Jung Kwon, Kook Hyung Kim, Cynthia Hemenway |
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| Abstract |
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The 3` region of Potato virus X (PVX) has the 74 nt 3`- nontranslated region (NTR) that is conserved among all potexviruses and contains several cis-acting elements for minus-strand and plus-strand RNA accumulation. Three stem-loop structures (SL1-SL3), especially formation of SL3 and U-rich sequence of SL2, and near upstream elements in the 3` NTR were previously demonstrated as important cis-acting elements. To investigate the binding of these cis-acting elements within 3` end with host protein, we used the electrophoretic mobility shift assays (EMSA) and UV-cross linking analysis. The EMSA with cellular extracts from tobacco and RNA transcripts corresponding to the 150 nt of the 3` end of PVX RNA showed that the 3` end of PVX formed complexes with cellular proteins. The specificity of protein binding was confirmed through competition assay by using with 50-fold excess of specific and non-specific probes. We also conducted EMSA with RNAs containing various mutants on those cis-acting elements (Δ10, SL3B, SL2A and Δ21; J Mol Biol 326, 701-720) required for efficient PVX RNA accumulation. These analyses supported that these cisacting elements are required for interaction with host protein(s). UV-cross linking analysis revealed that at least three major host proteins of about 28, 32, and 42 kDa in mass bound to these cis-elements. These results indicate that cis-acting elements from 3` end which are important for minus and plus-strand RNA accumulation are also required for host protein binding. |
| Key Words:
3` cis-elements, EMSA, host protein binding, PVX |
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