Summary
Procedures that were critical for crystallization of the glmS ribozyme-riboswitch RNA domain from the thermophilic Gram-positive bacterium Thermoanaerobacter tengcongensis are described. Experimental design based on screening multiple variant RNA sequences and techniques used to identify initial crystallization conditions were similar to those employed for most RNAs. However, serendipitous in-drop digestion of one RNA construct at a specific internucleotide linkage was crucial for the growth of high-quality glmS ribozyme crystals. Biochemical analysis of crystalline RNA identified the site of scission and guided design of an optimized bimolecular RNA construct. Finally, modifications of ionic strength and pH of solutions used for stabilization of the crystals were essential for optimal diffraction and binding of the activator glucosamine-6-phosphate, respectively. Although their details are specific to the glmS ribozyme, these general strategies may be useful for analyzing and improving crystals of other RNAs.
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Acknowledgments
A.R.F. is an Investigator of the Howard Hughes Medical Institute and was a Distinguished Young Scholar in Medical Research of the W.M. Keck Foundation. D.J.K. was a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-1863-05). This work was also supported by grants from the NIH (GM63576 to A.R.F. and GM084076 to D.J.K.) and the W.M. Keck Foundation.
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Klein, D.J., Ferré-D’Amaré, A.R. (2009). Crystallization of the glmS Ribozyme-Riboswitch . In: Serganov, A. (eds) Riboswitches. Methods in Molecular Biology, vol 540. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-558-9_10
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DOI: https://doi.org/10.1007/978-1-59745-558-9_10
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