Figure 4. Representative NMR spectra of the preQ1b riboswitch binding to its ligand. Arrows indicate characteristic changes in the spectra.
Figure 3. (a) Structure of riboswitch that controls expression of B. subtillis queCDEF gene. (b) Structure of preQ. (c,d,e,f) Predicted secondary structures of preQ1a-b, respectively. Species from which the riboswitch constructs were derived are listed prioer to each construct.
designed based on common binding rules for all SAM riboswitches, it may potentially target all three riboswitches, reducing the chances of bacteria developing antibiotic resistance. Since the structure of the SAM-I riboswitch was determined by another laboratory  and because the SAM-III riboswitch is narrowly distributed amongst bacteria , I focused my research on the SAM-II riboswitch. The preQ1 riboswitch recognizes preQ1, anintermediate in queuosine biosynthesis (Figure 3a, b) . Queuosine is a hypermodified nucleotide occupying the wobble position of the anticodon of certain transfer RNAs. The preQ1 riboswitch is located in the 5´ UTR of the queCDEF operon that is involved in preQ1 synthesis in a wide range of bacteria. Both the SAM-II and the preQ1 riboswitches have a common theme in their secondary structures. In contrast to other riboswitches, they are built around a stem-loop structure that may interact with the 3´ region of the riboswitch. Such secondary structures suggest a novel type of 3-D structure and metabolite binding in riboswitches, which may be important for drug design.
Materials and Methods In vitro transcription was chosen to generate large quantities of homogenous
SAM-II RNA for crystallization. DNA templates for in vitro transcription were prepared from chemically synthesized DNA oligonucleotides that were annealed together and cloned into a modified plasmid that carried the T7 promoter [18,19]. The plasmid was transformed into the XL1-Blue strain of E. coli, which was used in large scale plasmid DNA purification to generate preparative quantities of DNA for in vitro transcription reactions . PreQ1 RNA was short enough to be efficiently synthesized through chemical means and was purchased from Dharmacon. Riboswitch-ligand complexes were formed by mixing RNA with SAM or preQ1 in a buffer containing D2O and deuterated potassium acetate. The ligand was added to RNA to reach a 1 to 1 molar ratio while performing NMR spectroscopy until spectrum changes were no longer observed. For crystallization, each RNA-ligand complex was mixed with the same volume of reservoir solution (from commercial sparse matrix kits) and crystals were grown by vapor diffusion in either hanging or sitting drop formats . Crystals were shot by an X-ray beam with test frames recorded at three crystal orientations. Data sets for were also collected at the Advanced Photon Source (APS) (Chicago, IL).
The Stony Brook Young Investigators Review, Fall 2011
Results SAM-II constructs were designed for crystallization using published software to determine secondary structures in the riboswitch [21,22]. Two of the best-folding SAM-II riboswitches were chosen for the study. The first construct, 58 nucleotide long SAM-IIa, incorporates the central evolutionary conserved part of the sensing domain, while the second construct, 83 nucleotide long SAMIIb, contains a hairpin on the 3´ end of the sequence. The preQ1 riboswitch has the smallest metabolite-sensing domain, which, according to predictions , does not have a pronounced secondary structure. Four preQ1 RNA constructs, ranging from 34-57 nucleotides long, were designed for crystallization. NMR spectroscopy was used to monitor formation of complexes between riboswitches and their ligands in a 1 to 1 proportion. For example, the imino proton spectrum of free preQ1b RNA (Figure 4) shows four well resolved peaks and the addition of the ligand formed new peaks, representing changes in riboswitch base-pairing. Titration stopped after changes in the peaks were no longer observed. Formation of crystals requires macromolecules to be present in an oversaturated solution, which is achievable by a technique called vapor diffusion.