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| The EMBO Journal
(1999) 18, 3776–3782, doi:10.1093/emboj/18.13.3776 |
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| Assaying RNA chaperone activity in vivo using a novel RNA folding trap |
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| Elisabeth Clodi, Katharina Semrad and Renée Schroeder |
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Institute of Microbiology and Genetics, Vienna Biocenter, Dr Bohrgasse 9, A-1030 Vienna, Austria To whom correspondence should be addressed Renée Schroeder, renee@gem.univie.ac.at Received 16 November 1998; Revised 12 May 1999; Accepted 12 May 1999. |
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| Abstract |
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| In the absence of proteins, RNAs often misfold in vitro due to alternative base pairings which result from the molecule being trapped in inactive conformations. We identify an in vivo folding trap in the T4 phage td gene, caused by nine base pairs between a sequence element in the upstream exon of the td gene and another at the 3' end of the intron. During translation, the ribosome resolves this interaction; consequently the intron folds correctly and splicing occurs. The introduction of a stop codon upstream of this base pairing prevents resolution of the inactive structure so that splicing cannot proceed. We have used this folding trap to probe for RNA binding proteins which, when overexpressed, either resolve the misfolded structure or impede its formation in vivo. We distinguish between proteins which recognize the intron structure and those which bind non-specifically and apparently ignore the intron. The first class, e.g. Neurospora crassa CYT-18, can rescue the exonic trap and intron mutants which cause a structural defect. However, known RNA chaperones such as Escherichia coli StpA and S12 and the HIV protein NCp7, only resolve the exonic trap without suppressing intron mutations. Thus, this structural trap enables detection of RNA chaperone activity in vivo. |
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| Keywords: group I intron, RNA chaperone activity, RNA folding trap assay, splicing, T4 phage |
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