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

Hydroxyl radicals (. OH) can cleave the phosphodiester backbone of nuc leic acids and are valuable reagents in the study of nucleic acid stru cture and protein-nucleic acid interactions. Irradiation of solutions by high flux ''white light'' X-ray beams based on bending magnet beaml ines at the National Synchrotron Light Source (NSLS) yields sufficient concentrations of . OH so that quantitative nuclease protection (''fo otprinting'') 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 R NA to X-ray mediated . OH cleavage is equivalent. Both nucleic acids a re completely protected from synchrotron X-ray induced cleavage by the presence of thiourea in the sample solution, demonstrating that cleav age is suppressed by a free radical scavenger. The utility of this tim e-dependent approach to footprinting is demonstrated with a synchrotro n X-ray footprint of a protein-DNA complex and by a time-resolved foot printing analysis of the Mg2+-dependent folding of the Tetrahymena the rmophilia L-21 ScaI ribozyme RNA. Equilibrium titrations reveal differ ences among the ribozyme domains in the cooperativity of Mg2+-dependen t . OH protection. RNA . OH protection progress curves were obtained f or several regions of the ribozyme over timescales of 30 seconds to se veral minutes. Progress curves ranging from greater than or equal to 3 .5 to 0.4 min(-1) were obtained for the P4-P6 and P5 sub-domains and t he P3-P7 domain, respectively. The . OH protection progress curves har e been correlated with the available biochemical, structural and model ing data to generate a model of the ribozyme folding pathway. Rate dif ferences 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 conformatio n and protein-nucleic acid complexes. (C) 1997 Academic Press Limited.