The radiation responsive promoter of the Deinococcus radiodurans pprA gene

In a previous study, we identified a novel radiation-inducible protein PprA that plays a critical role in the radiation resistance of Deinococcus radiodurans [Narumi, I., Satoh, K., Cui, S., Funayama, T., Kitayama, S., Watanabe, H., 2004. PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation. Mol. Microbiol. 54, 278-285.]. Despite the interest in mechanisms underlying radiation responses in D. radiodurans, little is known about the radiation responsive promoter for radiation-inducible proteins. In this study, three transcriptional start points for pprA mRNA were identified by primer extension analysis, located at positions − 156, − 154 and − 22 upstream from the pprA translation initiation site. The amount of the three extended products increased in cells exposed to 2 kGy followed by a 0.5-h post-incubation. This suggested the existence of at least two radiation responsive promoters for pprA expression. Functional characterization of the upstream region of the pprA gene using a luciferase reporter assay revealed that the distal promoter is located between positions − 208 and − 156 from the translation initiation site, while the proximal promoter is located between positions − 57 and − 22. The region located between positions − 57 and − 38 was indispensable for proximal promoter activity. Site-directed mutagenesis of a thymine positioned at − 33 resulted in severe impairment of promoter activity, and suggested that the thymine functions as a master base for the proximal radiation responsive promoter. The product of the D. radiodurans pprI gene is thought to be a general switch in the radiation response [Hua, Y., Narumi, I., Gao, G., Tian, B., Satoh, K., Kitayama, S., Shen, B., 2003. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem. Biophys. Res. Commun. 306, 354-360.]. We examined the effect of pprI disruption on pprA promoter activity. The results suggested that up-regulation of pprA expression by the pprI gene product is triggered at the promoter level.