Characterization of regulatory roles of RprA sRNA in Escherichia coli

• Main Author: 蕭雅之
• Other Authors: Tseng, Ching-Ping
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/82908890955650287160

碩士 === 國立交通大學 === 生物科技學系 === 101 === Recent studies revealed that bacteria encode a tremendous number of small non-coding RNA (sRNA). In Escherichia coli, RprA is a 105 nt long sRNA. Previous studies have shown that RprA sRNA accumulates in the stationary phase in response to high osmolality. By base-pairing with the 5’-untranslated region of the target mRNA, it regulates the mRNA of rpoS, csgD, and ydaM in RNA stability or during protein translation. To investigate the regulatory role of RprA under high osmotic stress, a genome-wide transcriptome that overexpresses the rprA gene was compared with an rprA deletion strain using a microarray. The results were then confirmed via real-time PCR, and 10 genes were regulated using RprA sRNA in a time course, which suggests that they were direct targets of RprA sRNA. These genes are involved in carbon metabolism and membrane stress. The results indicate that RprA links the outer stimuli and central metabolism together to adjust cellular physiology in response to osmotic stress. The base-pairing site of RprA/target was predicted using RNAhybrid. The base-pairing site was tested with GFP translational fusion and was validated using rprA sequence deletion. We demonstrated that the lamB gene that encodes the phage receptor maltoporin is a novel and negatively controlled RprA target. By adapting these direct targets and their physiologic roles, we reconstructed the network governed by RprA sRNA to elucidate the mechanism of gene regulation in response to high osmolality.