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Nucleic Acids Research 2005 33(3):1058-1068; doi:10.1093/nar/gki237
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Published online 18 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Investigation of Mg2+- and temperature-dependent folding of the hairpin ribozyme by photo-crosslinking: effects of photo-crosslinker tether length and chemistry

Emily J. Borda1 and Snorri Th. Sigurdsson1,2,*

1 Department of Chemistry, University of Washington Seattle, WA 98195-1700, USA 2 University of Iceland, Science Institute Dunhaga 3, IS-107 Reykjavik, Iceland

*To whom correspondence should be addressed. Tel: +1 206 543 1610; Fax: +1 206 685 8665; Email: snorrisi{at}hi.is

Received August 1, 2004. Revised November 25, 2004. Accepted January 20, 2005.

We have used photo-crosslinking to investigate the structure and dynamics of four-way junction hairpin ribozyme constructs. Four phenylazide photo-crosslinkers were coupled to 2'-NH2-modified U+2 in the substrate and irradiated at different Mg2+ concentrations and temperatures. Consistent with the role of divalent metal ions in hairpin ribozyme folding, we observed more interdomain crosslinks in the presence of Mg2+ than in its absence. In general, we observed intradomain crosslinks to nucleotides 2–11 and interdomain crosslinks to the U1A binding loop. Crosslinks to A26 and G36 in domain B were also observed when crosslinking was carried out at –78°C. In contrast to crosslinking results at higher temperatures (0, 25 and 37°C), similar crosslinks were obtained in the presence and absence of Mg2+ at –78°C, suggesting Mg2+ stabilizes a low-energy hairpin ribozyme conformation. We also evaluated the effects of photo-crosslinker structure and mechanism on crosslinks. First, most crosslinks were to unpaired nucleotides. Second, shorter and longer photo-crosslinkers formed crosslinks to intradomain locations nearer to and farther from photo-crosslinker modification, respectively. Finally, fluorine substitutions on the phenylazide ring did not change the locations of crosslinks, but rather decreased crosslinking efficiency. These findings have implications for the use of phenylazide photo-crosslinkers in structural studies of RNA.


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