Structural analysis of putative transcriptional repressors from Streptomyces coelicolor A3(2) by the S-SAD method

Graduate School of Science, Hokkaido University* Department of Food and Bioscience, Faculty of Food and Nutrition, Beppu University**
○Kazuya Kondo* Takeshi Hayashi** Nobuhisa Watanabe* Isao Tanaka*

Streptomyces coelicolor A3(2) is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotic used in human and veterinary medicine. It is predicted that numerous regulatory genes are contained in the genome of S.coelicolor A3(2), and they are likely to adapt to a wide range of environmental condition. To reveal the regulatory mechanism of antibiotic and other useful secondary metabolite makes it possible to develop useful material production system by actinomyces.
In the genome of S.coelicolor A3(2), it is estimated that 150 putative TetR family regulatory proteins are encoded. They control gene expression of their products that are involved in multidrug resistance, enzymes implicated in catabolic pathway, biosynthesis of antibiotics and so on. These proteins have two domains; a C-terminal regulatory domain and an N-terminal DNA-binding domain which contains highly conserved helix-turn-helix motif. Despite of a high degree of sequence similarity at the DNA-binding domain, structural determination of those proteins by molecular replacement has difficulty for low similarity at the regulatory domain.
To elucidate the transcriptional mechanism, we attempt the S-SAD method, which means Single wavelength Anomalous Diffraction method using Sulfur atoms in the protein as anomalous scatterers, for analyzing crystal structure of transcription factors. Our targets are putative TetR family regulatory proteins; SCO0241, SCO0332, SCO7518, SCO7651 and SCO7815, and their ΔF/F value at Cr/Cu Ka radiation are expected as 1.653/0.806, 1.045/0.509, 1.086/0.529, 1.221/0.595 and 1.425/0.694 by those sulfur contents, respectively. In this conference, we'll discuss the results of in-house S-SAD method for structural determinations.