Assaying RNA chaperone activity in vivo in bacteria using a ribozyme folding trap

Here, we report an assay to evaluate the intracellula r RNA chaperone activity of a protein of interest in vivo in bacterial cells. Themethod is based on self-splicing of the group I intron, which is located in the thymidylate synthase (td) gene of phage T4. A previouslydescribed td mutant (tdSH1) has significantly impaired splicing due to formation o f splicing-incompetent alternative structures. In thisprocedure, overexpression of RNA chaperones in the presence of the td mutant SH1 is used to evaluate whether the putative RNAchaperoneisabletorescuetheincorrectlyfoldedgroupIintron.TheabilityoftheRNAchaperonetoassistduringfoldingismeasuredindirectly by assessing the difference between the splicing efficiencies of the td mutant in the absence and in the presence of the RNAchaperone. This procedure can be completed in 5–6 d, not inc luding the time needed to clone the putative RNA chaperone.INTRODUCTIONRNA molecules have a great flexibility and easily become trappedin misfolded conformations—a phenomenon known as the RNAfolding problem