Small noncoding RNAs as multipotent components for regulation of cellular metabolism

Abstract

Now, it is undoubted that noncoding RNA has been deeply involved in various metabolic pathways in many different organisms. Although nearly 100 small noncoding RNAs (sRNAs) have been experimentally verified in E. coli, it is still not known how many sRNAs are present in that bacterium. Extensive studies during the past decade revealed that bacterial sRNAs participate in variety of cellular metabolic pathways as a key regulator. Knowledge about the roles of bacterial sRNAs has been continuously expanded through finding not only functions of newly identified sRNAs but also new roles of even previously known sRNAs. Considering the number of identified sRNAs and their known functions until now, what we have known about functions of sRNAs seems to be very limited. Therefore, there are many, yet-to-be-discovered, roles of sRNAs in cellular processes including biofilm formation, antibiotics resistance, and virulence. In this thesis, novel functions of sRNAs were investigated by characterizing sRNA interactomes with protein targets or another RNA target, and by observing phenotypic changes of bacteria, such as biofilm formation and enhanced resistance against acid. Novel sRNA interactomes in E. coli were identified by using a Yeast Three-hybrid system and affinity pull-down, and by designing a bioinformatical target prediction protocol. An E. coli ORF library was constructed on plasmid pJG4-5Sfi and the Yeast Three-hybrid system has been optimized with the library. The optimized system was successfully applied to identify new interactions between sRNA and proteins. Unexpectedly, a DNA binding protein, XylR, was characterized as a novel RNA binding protein, implying that there might be an auto-regulation mechanism in XylR-mediated regulation. In addition, an RNA expression vector, pRT3, was developed for efficient discrimination of RNA-independent false positives in the Yeast Three-hybrid system. A plasmid library expressing 99 experimentally verified E. coli sRNAs was...