Abstract
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 to 100 ms. 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 suppressed by a free radical scavenger. The utility of this time-dependent approach to footprinting is demonstrated with a synchrotron X-ray footprint of a protein-DNA complex and by a time-resolved footprinting analysis of the Mg2+-dependent folding of the Tetrahymena thermophilia L-21 ScaI ribozyme RNA. Equilibrium titrations reveal differences among the ribozyme domains in the cooperativity of Mg2+-dependent . .OH protection. RNA . .OH protection progress curves were obtained for several regions of the ribozyme over timescales of 30 seconds to several minutes. Progress curves ranging from ≥3.5 to 0.4 min–1 were obtained for the P4-P6 and P5 sub-domains and the P3-P7 domain, respectively. The . .OH protection progress curves have been correlated with the available biochemical, structural and modeling data to generate a model of the ribozyme folding pathway. Rate differences observed for specific regions within domains provide evidence for steps in the folding pathway not previously observed. 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.
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More From: Biochemical and Biophysical Research Communications
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