RNA Catalytic Activity as a Probe of Chaperone-Mediated RNA Folding

For structured RNAs that possess catalytic activity, this activity provides a powerful probe for measuring the progress of folding and the effects of RNA chaperone proteins on the folding rate. The crux of this approach is that only the natively folded RNA is able to perform the catalytic reaction. This method can provide a quantitative measure of the fraction of native RNA over time, and it can readily distinguish the native state from all misfolded conformations. Here we describe an activity-based method measuring native folding of ribozymes derived from self-splicing group I introns, and we show how the assay can be used to monitor acceleration of native folding by DEAD-box RNA helicase proteins that function as general RNA chaperones. By measuring the amount of substrate that is converted to product in a rapid first turnover, we describe how to determine the fraction of the ribozyme population that is present in the native state. Further, we describe how to perform a two-stage or discontinuous assay in which folding proceeds in stage one and then solution conditions are changed in stage two to permit catalytic activity and block further folding. This protocol allows folding to be followed under a broad range of solution conditions, including those that do not support catalytic activity, and facilitates studies of chaperone proteins.