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.
Ohba
Hirofumi
H
Research Group for Biotechnology Development, Department of Ion-beam-applied Biology, Japan Atomic Energy Research Institute, 1233 Watanuki-machi, Takasaki, Gunma 370-1292, Japan.
Satoh
Katsuya
K
Yanagisawa
Tadashi
T
Narumi
Issay
I
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D034301:Deinococcus / Q000235:genetics / Q000528:radiation effects*; D011401:Promoter Regions, Genetic / Q000528:radiation effects*; D011836:Radiation Tolerance / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers