CHESS DMR Filming RNA transcription at atomic resolution Joel Brock, Cornell University, DMR Structure of the transcript initiation complex. B)schematic of the transcript initiation. C) sequence of the P2_7c DNA used for crystallization with binding sites indicated (gray box shows disordered crystal structures). Basu R. S., Murakami K. S. (2013) Watching the bacteriophage N4 RNA polymerase transcription by time-dependent soak-trigger-freeze X-ray crystallography. J Biol Chem. 288(5), Intellectual Merit: RNA polymerase (RNAP) is the molecular Xerox machine that mediates information transfer from DNA to ribosomes for protein synthesis. Usually DNA and RNA polymerases are complex structures that require the presence of multiple protein co-factors. In this study Basu and Murakami used the CHESS F1 end station to dissect the reaction mechanism of a single subunit viral RNAP via a time- resolved soak-trigger-freeze X-ray crystallography method. Their work provides clues to the two-metal-ion reaction mechanism that was first proposed two decades ago by Steitz and Steitz. They first soak crystals of RNAP-promoter DNA complex with metal ions and nucleotides, followed by flash-cooling the crystals at different time points. By doing this they are able to track the appearance and disappearance of electron density corresponding to incoming or outgoing metal ions, and assign conformational changes of the catalytically essential O-helix to specific events in the reaction scheme.

CHESS DMR Structure of the transcript initiation complex. The amino acid residues involved in nucleotides and metal binding are shown. Mn2+ and water is depicted by yellow and cyan spheres, respectively. Basu R. S., Murakami K. S. (2013) Watching the bacteriophage N4 RNA polymerase transcription by time-dependent soak-trigger-freeze X-ray crystallography. J Biol Chem. 288(5), Broader Impacts: A major challenge for structural biology is to understand atomic level macromolecular motions during enzymatic reactions. X-ray crystal- lography can reveal high resolution structures, but usually only static views. These researchers used time- dependent soak-trigger-freeze X-ray crystallography, soaking nucleotide and divalent metal into the bacteriophage RNA polymerase (RNAP)-promoter DNA complex crystals to trigger the nucleotidyl transfer reaction, then they freeze crystals at different time points to capture real-time intermediates in the pathway of transcription. In each crystal structure, different electron density maps corresponding to the nucleotide and metal were revealed at the RNAP active site which allows watching the nucleotide and metal bindings and the phosphodiester bond formation in a time perspective. They captured elusive intermediates exploiting the slow enzymatic reaction in crystallo, demonstrating that simple time-dependent soak-trigger-freeze offers a direct means for monitoring enzymatic reactions. Filming RNA transcription at atomic resolution Joel Brock, Cornell University, DMR

CHESS DMR Near atomic resolution crystal structure of Ath α-DOX. Extended inserts of Ath α-DOX; view of the ionic interactions between the two inserts that serve to stabilize and rigidify the loops. Goulah C. C., Zhu G., Koszelak-Rosenblum M., Malkowski MG. (2013) The crystal structure of α-Dioxygenase provides insight into diversity in the cyclooxygenase-peroxidase superfamily. Biochemistry. 52(8), Intellectual Merit: Unlike humans, plants lack the immune system required to fend off attacks from pathogens. Plants rely on a nonspecific response to deal with an outside attack (in humans, this type of response is known as innate immunity). Examples of plant defense mechanisms include the production of pathogen-degrading enzymes, the production of chemicals (sometimes toxic), and deliberate cell suicide. In a recent study by the group of structural biology professor Michael Malkowski of SUNY Buffalo, new light was shed on the reaction mechanism and substrate specificity of an enzyme that belongs to the pathogen inducible oxygenase family. As part of the host defense response in Arabidopsis thaliana, levels of the heme-containaing α-Dioxygenase (Ath α-DOX) are up-regulated to produce oxygenated fatty acids, chemicals which are often toxic to pathogens. Malkowski and coworkers report a high resolution crystal structure of Ath α-DOX, the first crystal structure of its kind. Shedding light on how plants defend themselves Joel Brock, Cornell University, DMR

CHESS DMR Substrate binding and active site. Close-up of the active site residues in Ath α-DOX. Goulah C. C., Zhu G., Koszelak-Rosenblum M., Malkowski MG. (2013) The crystal structure of α-Dioxygenase provides insight into diversity in the cyclooxygenase-peroxidase superfamily. Biochemistry. 52(8), Broader Impacts: Unlike humans, plants lack the immune system required to fend off attacks from pathogens but instead rely on a nonspecific response to deal with an outside attack. The structure of Ath α- DOX presented here provides the first structural snapshot of a member of the α-DOX subfamily within the peroxidase-cyclooxygenase superfamily. While LOX is found in both plant and animal kingdoms, no protein with cyclooxygenase activity has been identified in plants. In 1998, Castresana and colleagues identified a protein in tobacco leaves and Arabidopsis that was upregulated during the host defense response. This subfamily is part of the superfamily whose members developed the ability early in the evolutionary process to generate oxidants as a general defense strategy. Characterization of members of this superfamily has provided insight into evolutionary relationships and allowed for critical comparisons to be made between proteins in different subfamilies. Shedding light on how plants defend themselves Joel Brock, Cornell University, DMR