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Time-resolved synchrotron X-ray "footprinting", a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding.

TitleTime-resolved synchrotron X-ray "footprinting", a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding.
Publication TypeJournal Article
Year of Publication1997
AuthorsSclavi, B, Woodson, S, Sullivan, M, Chance, MR, Brenowitz, M
JournalJ Mol Biol
Date Published1997 Feb 14
KeywordsAnimals, Bacterial Proteins, Base Sequence, Crystallography, X-Ray, DNA, Escherichia coli, Escherichia coli Proteins, Hydroxides, Kinetics, Lac Repressors, Magnesium, Molecular Sequence Data, Nucleic Acid Conformation, Protein Conformation, Proteins, Repressor Proteins, RNA, RNA, Catalytic, Synchrotrons, Tetrahymena thermophila

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 Mg(2+)-dependent folding of the Tetrahymena thermophilia L-21 ScaI ribozyme RNA. Equilibrium titrations reveal differences among the ribozyme domains in the cooperativity of Mg(2+)-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 > or = 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.

Alternate JournalJ. Mol. Biol.
PubMed ID9054977
Grant ListGM39929 / GM / NIGMS NIH HHS / United States
GM51506 / GM / NIGMS NIH HHS / United States
GM52348 / GM / NIGMS NIH HHS / United States