22] Time-resolved synchrotron X-ray footprinting and its application to RNA folding

Abstract

This chapter discusses the application of synchrotron X-ray footprinting for studying RNA folding at beam line X-9A. Beam line X-9A is a bending magnet beam line producing white light over an energy range of 3–30 keV. The radiolysis reaction products are separated using polyacrylamide gel electrophoresis (PAGE). A storage phosphor screen and associated imager are used to acquire a digital image of the electrophoretogram for densitometric analysis. Band intensity is quantitated using the molecular dynamics image quant or equivalent image analysis software. The intensity within a protected region and a reference region is quantitated for each lane on the gel. The reference region is a set of bands that accounts for variability in sample loading in the individual lanes but that shows the same degree of protection throughout the folding process. The densitometric results are exported to a spreadsheet (Microsoft Excel) for further processing and analysis. Each protected region is divided by the corresponding reference region in the same lane. The resulting data points are plotted as a function of reaction time. The protection quantitated to obtain this curve represents bases 118–120 within the P4–P6 domain of the ribozyme and appears at a rate 0.2 sec −1 . This protection is believed to represent a tertiary contact with bases within the peripheral domain P9.1 that show a comparable rate of protection.