Investigation of ribozyme structure and dynamics through photochemical crosslinking and metal ion cleavage.

摘要

Part I of this dissertation describes the use phenylazide crosslinkers to study changes in structure and dynamics of the hammerhead and hairpin ribozymes as a function of Mg2+ concentration, to investigate the effects of temperature on the structure and dynamics of the hairpin ribozyme, and to investigate the effects of crosslinker tether lengths and photoreactive mechanisms on the extent and locations of crosslinks in both ribozymes. Our Mg2+-dependence results suggest the hammerhead ribozyme is more flexible than the hairpin ribozyme, and our temperature-dependence results suggest Mg2+ stabilizes a low energy conformation of the hairpin ribozyme. Finally, we found that unsubstituted photocrosslinkers generated a higher number of crosslinks in higher yields than their tetrafluoro-substituted counterparts.; Part II describes the use of metal ion cleavage to study the structure and dynamics of the hammerhead ribozyme, as well as its interactions with antibiotics. We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. This new cleavage site has an unusual pH dependence that points toward a pH-dependent conformational change in the hammerhead ribozyme. We have previously described Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes.; Finally, we have investigated the interactions of two antibiotics, neomycin B and chlortetracycline (CTC), with the hammerhead ribozyme using the U4 and A9 Zn2+ cleavage sites. Our results suggest that the inhibitory mechanism of neomycin does not include competition with the metal ion bound to the A9/G10.1 metal-ion binding site, as previously proposed, and that there is either more than one neomycin binding site or multiple binding modes at a single site in the hammerhead ribozyme. Furthermore, the accessibilities and/or affinities of disparate neomycin binding sites or binding modes are dependent on the ionic strength and the pH of the medium.