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REPORTS for 3-D structure determination by X-ray crystallography, signifying that the obtained X-ray data may help determine the 3-D structure of the preQ1 riboswitch. Further analysis of crystal data suggested stacking conformations of RNA-ligand complexes that were different from previously observed stacking interactions for non-methylated RNA. This suggests that crystallization of RNA with 2’-O-methylated nucleotides generates hydrophobic patches, reorganizing RNA in different conformations. 2’-O-methylations can also be used to acquire phase information for structure determination by replacing the oxygen with a selenium atom. Such a substitution would permit application of single- and multiple-wavelength anomalous dispersion (SAD and MAD) techniques to the selenium atom, simplifying the daunting task of 3-D structure determination.

Conclusions As a unique RNA system for gene expression control, riboswitches stand out as potential targets for antimicrobial drugs because they control essential genes in a wide range of bacteria and can recognize small drug-like molecules. With the help of 3-D structures, these natural metabolites may be redesigned to modern drug standards. The riboswitch-ligand complexes for both SAM-II and preQ1 were successfully crystallized, completing the primary goal of the project. Careful optimization of crystal conditions led to the generation of preQ1 crystals that diffracted at 3.1 Å, a resolution suitable for determination of the 3-D structure. Additional work on the preQ1 riboswitch will likely reveal novel metabolite binding rules, which may contribute to the design of future riboswitch-targeting antimicrobial drugs.

References

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Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 89-108 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2006). 4. Nudler, E. & Mironov, A.S. The riboswitch control of bacterial metabolism. Trends Biochem. Sci. 29, 11-17 (2004). 5. Blount, K.F. & Breaker, R.R. Riboswitches as antibacterial drug targets. Nat. Biotechnol. 24, 1558-1564 (2006). 6. Dann, C.E., 3rd et al. Structure and mechanism of a metal-sensing regulatory RNA. Cell 130, 878-892 (2007). 7. Sudarsan, N. et al. Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine. Chem. Biol. 12, 1325-1335 (2005). 8. Blount, K.F. et al. Antibacterial lysine analogs that target lysine riboswitches. Nat. Chem. Biol. 3, 44-49 (2007). 9. Sudarsan, N. et al. An mRNA structure in bacteria that controls gene expression by binding lysine. Genes Dev. 17, 26882697 (2003). 10. Wakeman, C.A. et al. Structural features of metabolite-sensing riboswitches. Trends Biochem. Sci. 32, 415-424 (2007). 11. Corbino, K.A. et al. Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria. Genome Biol. 6, R70 (2005). 12. Epshtein, V. et al. The riboswitchmediated control of sulfur metabolism in bacteria. Proc. Natl. Acad. Sci. U S A 100, 5052-5056 (2003). 13. Fuchs, R.T. et al. The SMK box is a new SAM-binding RNA for translational regulation of SAM synthetase. Nat. Struct. Mol. Biol. 13, 226-233 (2006). 14. McDaniel, B.A. et al. Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA. Proc. Natl. Acad. Sci. U S A 100, 3083-3088 (2003). 15. Winkler, W.C. et al. An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat. Struct. Biol. 10, 701-707 (2003). 16. Montange, R.K. & Batey, R.T. Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441, 1172-1175 (2006). 17. Roth, A. et al. A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain. Nat. Struct. Mol. Biol. 14, 308-317 (2007).

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18. Sambrook, J. et al. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989). 19. Vieira, J. & Messing, J. Production of single-stranded plasmid DNA. Methods Enzymol. 153, 3-11 (1987). 20. McPherson, A. Introduction to Macromolecular Crystallography (WileyLiss, Wilmington, 2003). 21. Griffiths-Jones, S. et al. Rfam: annotating non-coding RNAs in complete genomes. Nucleic. Acids. Res. 33, D121124 (2005). 22. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 34063415 (2003). 23. Price, S.R. et al. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J. Mol. Biol. 249, 398-408 (1995). 24. Klepper, F. et al. Robust Synthesis and Crystal-Structure Analysis of 7-Cyano-7-deazaguanine (PreQ0 Base) and 7-(Aminomethyl)-7-deazaguanine (PreQ1 Base). Helvetica Chimica Acta 88, 2610-2616 (2005). 25. COLLABORATIVE COMPUTATIONAL PROJECT. The CCP4 suite: programs for protein crystallography. Acta Cryst. D 50, 760-763 (1994). 26. Schwalbe, H. et al. Structures of RNA switches: insight into molecular recognition and tertiary structure. Angew. Chem. Int .Ed Engl. 46, 1212-1219 (2007).

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