Assaying RNA chaperone activity in vivo using a novel RNA folding trap

In the absence of proteins, RNAs often misfold in vitro due to alternative base pairings which result from the molecule being trapped in inactive conf ormations. We identify an in vivo folding trap in the T4 phage td gene, cau sed by nine base pairs between a sequence element in the upstream exon of t he td gene and another at the 3' end of the intron, During translation, the ribosome resolves this interaction; consequently the intron folds correctl y and splicing occurs. The introduction of a stop codon upstream of this ba se pairing prevents resolution of the inactive structure so that splicing c annot proceed. We have used this folding trap to probe for RNA binding prot eins which, when overexpressed, either resolve the misfolded structure or i mpede its formation in vivo. We distinguish between proteins which recogniz e the intron structure and those which bind non-specifically and apparently ignore the intron, The first class, e.g, Neurospora crassa CYT-18, can res cue the exonic trap and intron mutants which cause a structural defect. How ever, known RNA chaperones such as Escherichia coli StpA and S12 and the HI V protein NCp7, only resolve the exonic trap without suppressing intron mut ations. Thus, this structural trap enables detection of RNA chaperone activ ity in vivo.