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Simon Hall 320D

Charles E. Dann III was awarded a B.S., ACS, degree in chemistry from Millsaps College in Jackson, Mississippi, in 1996. He received his Ph.D. from the Department of Biophysics & Biophysical Chemistry at Johns Hopkins University School of Medicine in Baltimore, Maryland, while working in the laboratory of Daniel Leahy. In 2002, Dr. Dann was invited to continue his studies at the University of Texas Southwestern Medical Center at Dallas as a postdoctoral fellow under the supervision of Nobel Laureate Johann Deisenhofer. After a year in this position, he received the Sara and Frank McKnight Fellowship to work as an independent fellow. Dr. Dann held this position until joining the faculty in the Department of Chemistry at Indiana University in August 2008.

The research in Dr. Dann’s laboratory focuses on determining atomic resolution structures of RNA and protein macromolecules by X-ray crystallography. An emphasis is placed on cis-acting regulatory RNAs termed riboswitches and the gene products (proteins) that they regulate. In addition to structural biology, we are interested in both manipulating riboswitches to generate engineered molecular sensors (on/off switches) and testing riboswitches as targets in small molecule screens to discover new antibiotics. Projects in the laboratory afford the researcher opportunities to learn computational modeling, structural and biophysical techniques as well as general biochemistry and molecular biology in both RNA and protein systems.

Research

Our lab is focused on the characterization of riboswitches, structured cis-acting RNA elements in messenger transcripts (mRNAs). Most riboswitches sense key metabolites, and this sensing event results in genetic regulation most commonly through changes in transcriptional termination or translation initiation. We use a combination of biochemical and biophysical techniques to probe the mechanism of action of riboswitches and proteins with an emphasis on X-ray crystallography to determine atomic resolution models of the macromolecule of interest (i.e. RNA, protein, or complexes).

1) X-ray Crystallography of Riboswitches. This focus of the laboratory is aimed at understanding regulation by riboswitches at the biochemical and ultimately at the atomic level via the use of X-ray crystallography. We have recently characterized the M-box aptamer domain, previously referred to as the ykoK orphan riboswitch, and shown that it functions as a Mg²⁺-sensing riboswitch. Our in vivo and in vitro data show that the Bacillus subtilis ykoK gene transcript contains a structured cis-acting RNA element that responds to divalent ion levels. In vivo expression studies show that this RNA element is required for downregulation of its gene products in the presence of normal levels of Mg²⁺. To elucidate its molecular mechanism, the structure of the M-box aptamer was determined by X-ray crystallography (Figure 1). The structure contains six Mg²⁺, four of which are in a region of tertiary contacts consisting of nucleotides that are highly conserved and form a complex set of contacts that regulation transcriptional termination. This study illustrates a molecular mechanism by which RNA can sense metal ions for the purpose of gene regulation. Though the characterization of this particular RNA is nearing completion, we are pursuing both biochemical and structural studies on a number of additional unique riboswitches that bind to a variety of effector molecules.

2) – Biochemical and Structural Characterization of Proteins Regulated by Riboswitches. The information obtained by studying riboswitches can often lend insight into the function of its regulated gene products. In particular, we are looking for riboswitch-regulated proteins that have roles in microbial pathogenicity. To this end, we are searching pathogenicity islands in bacteria for the presence of riboswitches with regulated proteins of unclear function that could benefit from biochemical and structural analyses. Based on the particular class of riboswitch regulating a given protein, we can infer and subsequently test candidate functions for the protein of interest. Using this approach, we have ascribed a clear function to a poorly understood, yet potentially disease relevant protein in Mycoplasma hyorhinis. Subesequent gel probing and isothermal titration calorimetry experiments have verified candidate ligand binding to both the riboswitch and the protein, respectively. In addition, we have determined the structure of the protein bound to its ligand by X-ray crystallography to 1.6 Å using sulfur SAD (Figure 3). Using these data, we are now poised to look into the mechanisms by which this protein may promote pathogenesis by altering levels of its ligand. This ongoing work demonstrates the method by which we will select protein targets for additional structure determination projects.

3) Manipulating and Targeting Riboswitches. As riboswitches are relatively small macromolecules (~15 – 100 kDa) that are capable of binding with nanomolar affinity and high selectivity to small molecule ligands, we believe that these RNAs can be engineered as in vivo sensors for their natural ligands and potentially targeted by small molecules. We are developing methodologies in the laboratory to create both prokaryotic and eukaryotic riboswitch-based sensors for key metabolites including S-adenosylmethionine (SAM), flavin mononuclotide (FMN), adenosylcobalamin (vitamin B12), and thamine pyrophosphate (TPP). Taken one step further, we would like to engineer a riboswitch capable of binding a non-biologically relevant small molecule to use as a molecular on/off switch for temporal control of gene expression (i.e. an inducible promoter). Lastly, as riboswitches control the levels of key metabolite pools via the genes they regulate, we are testing whether riboswitches are feasible candidates for small molecule screening for effector molecules that could act as antibiotics.

Publications

Dann, C.E. III, Wakeman, C.A., Sieling, C.L., Baker, S.C., Irnov, I., and Winkler, W.C. "Structure and Mechanism of a Metal-Sensing Regulatory RNA." Cell 130:5, 878-92, 2007.

Wakeman, C.A., Winkler, W.C., and Dann, C.E. III. "Structural Features of Metabolite-Sensing Riboswitches." Trends Biochem Sci 32:9, 415-24, 2007.

Dann, C.E. III and Winkler, W.C. "RNA allostery glimpsed." Nat Struct Mol Biol 13:7, 569-71, 2006 (News and Views).

Dann, C. E. III, Bruick, R. K. "Dioxygenases as O2-dependent regulators of the hypoxic response pathway." Biochem Biophys Res Commun 338:1, 639-47, 2005 (review).

Shah, S., Lee, S. F., Tabuchi, K., Hao, Y. H., Yu, C., LaPlant, Q., Ball, H., Dann, C. E. III, Sudhof, T., Yu, G. "Nicastrin functions as a gamma-secretase-substrate receptor." Cell 122:3, 435-47, 2005.

Hamaoka, B., Dann, C.E. III, Geisbrecht, B.V., and Leahy, D.J. "Crystal structure of Caenorhabditis elegans HER-1 and characterization of the interaction between HER-1 and TRA 2A." Proc Natl Acad Sci U S A 101:32, 11673-11678, 2004.

Dann, C. E. III, Bruick, R. K. and Deisenhofer, J. "Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway." Proc Natl Acad Sci U S A 99:24, 15351-6, 2002.

Dann, C.E. III, Hsieh, J.C., Rattner, A.., Sharma, D., Nathans, J. and Leahy, D.J. "Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains." Nature 412, 86-90, 2001.

Ooi, S.L., Dann, C. III, Nam, K., Leahy, D.J., Dahma, M.J., and Boeke, J.D. "Ribonucleases, Part A: Functional Roles and Mechanisms of Action. 'RNA lariat debranching enzyme.'" Methods in Enzymology 341, 2001.

Leahy, D.J., Dann, C.E. III,Longo, P.,Perman, B., and Ramyar, K. "A mammalian expression vector for expression and purification of secreted proteins for structural studies." Protein Expression and Purification 20:3, 500-506, 2000.

Ward, T.J., Dann, C.E. III, Brown, A.P. "Separation of enantiomers using vancomycin in a countercurrent process by suppression of electroosmosis." Chirality 8:1, 77-83, 1996.

Ward, T.J., Dann, C.E. III, Blaylock, A. "Enantiomeric resolution using the macrocyclic antibiotics rifamycin B and rifamycin SV as chiral selectors for capillary electrophoresis." Journal of Chromatography A 715, 337-344, 1995.

Awards

• Rigaku Post-Doc Travel Bursary Award, 2007
• Finalist, Burroughs Wellcome Career Award in the Biomedical Sciences, 2005
• Sara and Frank McKnight Independent Fellowship, 2003-2008
• Phi Beta Kappa, Millsaps College, 1996

Highlights