TIME RESOLVED SYNCHROTRON XRAY "FOOTPRINTING"

Hydroxyl radicals (OH) can cleave the phosphodiester backbone of nucleic acids and are valuable reagents in the study of nucleic acid structure and protein-nucleic acid interactions. Irradiation of solutions by high flux "white light" x-ray beams based on bending magnet beamlines at the National Synchrotron Light Source (NSLS) yields sufficient concentrations of OH so that quantitative nuclease protection ("footprinting") studies of DNA and RNA can be conducted with a duration of exposure in the range of 50 - 100 msec. The sensitivity of DNA and RNA to x-ray mediated OH cleavage is equivalent. Both nucleic acids are completely protected from synchrotron x-ray induced cleavage by the presence of thiourea in the sample solution, demonstrating that cleavage is mediated exclusively by free radicals. The utility of this time-dependent approach to "footprinting" is demonstrated by obtaining a synchrotron x-ray "footprint" of a protein-DNA complex and by a time-resolved "footprinting" analyses of the Mg2+-dependent folding of the Tetrahymena thermophilia L-21 Sca I ribozyme RNA. Equilibrium titrations reveal differences among the ribozyme domains in the cooperativity of Mg2+-dependent folding. RNA folding progress curves over timescales of 30 sec. to several minutes were obtained for several regions of the ribozyme. The rate constants obtained for the P3-P7, P4-P6 and P5 domains are comparable with those obtained by Zarrinkar & Williamson [Science 265: 918-924, 1994] using a hybridization-competition assay and confirm the hierarchy of the folding of the ribozyme domains established by these investigators. The folding rates obtained for individual regions within domains suggest additional steps in the folding pathway. Synchrotron x-ray "footprinting" is a new approach of general applicability for the study of time-resolved structural changes of nucleic acid conformation and protein-nucleic acid complexes.