The Regulation Mechanism Of Sig1 Factor In Deinococcus Radiodurans R1 Involved In Heat Shock Response

The Extra-Cytoplasmic Function sigma factors（ECF-σ）, are the most diverse part among σ factors that play an important role in protecting the organism from the environmental stresses by its regulation as the response. Deinococcus radiodurans R1 has stronger resistance and excellent adaptability against irradiation, oxidation and desiccation. D. radiodurans R1 contains three sigma factors, SigA/RpoD（DR₀916）, Sig1（DR₀180） and Sig2（DR₀804） and only the Sig1 belongs to the ECF-σ family. The sig1 mutant displayed high sensitive to oxidation and heat stress, which causes great growth deficiency, especially under the heat stress. Few study showed that the some heat-related genes expression decreased in sig1 mutant, but little is known about the regulatory mechanism of Sig1 under heat shock. This article studied the regulation of Sig1 to its target genes using transcriptional files, qPCR, β-galactosidase and gel shift assay. The main results obtained as follows.1. We performed comparative transcriptional analysis of D. radiodurans R1 and sig1 mutant after a temperature elevated from 30℃ to 48℃ for 2h. In total, 674 genes were up-regulated and 707 genes were down-regulated in D. radiodurans R1 under heat shock. These genes including the pathway of high temperature perception, signal transduction and response. There are 90 genes up-regulated and 980 genes down-regulated in sig1 mutant under heat shock, which involved in the synthesis of ribosome, purine, pyrimidine and amino acid metabolism and stress-related pathways. For example, 22 main subunits of ribosome in sig1 mutant displayed significant expression differences compared to the wild type under 48℃ for 2 hours. The expression of rpmD, rpmJ, rpsO, rpsH, rpmF and rpmG genes decreased above 2 fold（p<0.05）, which has an effect on mRNA synthesis, and also, the expression of 22 genes in purine metabolism and 10 genes in pyrimidine metabolism decreased evidently impeding the transcription process. In addition, amino acid synthesis, fatty acid synthesis and other pathways related genes are influenced inhibiting the growth of sig1 mutant. The heat shock related genes such as hsp20,hslJ and dnaJ1 down-regulated 2 fold verified by qPCR. Other stress-related genes including csp, pspA and fur were greatly down-regulated over 3 fold（p<0.05） and DNA repair-associated genes, recQ and ruvA, are also down-regulated significantly. Transcriptional analysis approved that Sig1 plays the global regulation role under heat shock.2. Sig1 belonging to ECF-σ factor contains conserved DNA-binding domain region 2 and region 4 based on homology modeling. Both two regions can bind to the target gene’s promoter-10 Box（region 2） and-35（region 4） Box specifically. These 266 genes expression, induced in D. radiodurans R1, were decreased in sig1 mutant under heat shock. We took an analysis of the down-regulated genes promoter by Softberry and Virtual Footprint software, which revealed that there are consensus sequence in-10 Box and-35 Box. So we speculated that the-10 Box is 5’-CGTAAAC-3’ and-35 Box is 5’-（T）T（G）AC（T）-3’.3. β-galactosidase assay by fusing the promoter（containing-10 Box and-35Box） of the down-regulated genes hsp20（encoding small heat shock protein）, csp（encoding cold shock protein）, pspA（encoding membrane integral protein） and fur（encoding ferric uptake regulation protein） to the lacZ reporter gene identified the regulation of Sig1 under heat shock. Furthermore, we took an extensive research about the small heat shock protein Hsp20. It’s highly induced in the D. radiodurans R1 about 7 fold, and decreased in sig1 mutant about 3 fold to the wild type. The survival curve showed that the hsp20 mutant is sensitive to heat stress with 3 order of magnitudes decline. In addition, the gel shift assay（EMSA） verified the interaction between the Sig1 and hsp20 promoter. These results indicated that Sig1 can regulate the hsp20, csp, pspA and fur expression under heat shock directly.This dissertation revealed that Sig1 can interact with the hsp20 promoter and involved in the synthesis of ribosome, purine, pyrimidine and heat stress regulation, which provides theoretical basis for the further work of heat shock response mechanism.