Artificial sRNA: an efficient approach for exploring sRNA-mediated gene silencing mechanisms in Escherichia coli = 인공 sRNA: 대장균에서 sRNA-매개 유전자 침묵 메커니즘 탐구를 위한 효율적인 접근 방법

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Non coding RNA (ncRNA) is a class of RNAs that function without being translated into protein. Above all, antisense RNA is a type of ncRNA that complementarily binds with mRNA to regulate its translation. But, all experimental papers have been confined to sRNA’s specific target region and whole mRNA region scanning has not been done and there have never been efficiently controlled study considering their antisense RNA’s structure. Therefore an artificial small RNA (afsRNA) scaffold which was designed from an Escherichia coli sRNA, SibC was made. Using the lacZ reporter system, the gene silencing effects of afsRNAs were examined to explore the sRNA-mediated gene silencing mechanisms in E. coli. Substitution of the original target recognition sequence with a new sequence recognizing lacZ mRNA led to effective reduction of lacZ gene expression. Single-strandedness of the target recognition sequences in the scaffold was essential for effective gene silencing. The target recognition sequence was shortened to 10 nt without significant loss of gene silencing, although this minimal length was limited to a specific target mRNA sequence. In cases where afsRNAs had mismatched (forming internal loops) or unmatched (forming bulges) regions in the middle of the target recognition sequence, internal loop-forming afsRNAs were more effective in gene silencing than those that formed bulges. Unexpectedly, gene silencing by afsRNA was not decreased, but increased upon hfq disruption in E. coli, particularly when interactions between afsRNA and mRNA were weak, suggesting that Hfq is possibly involved in destabilization of the RNA-RNA duplex, rather than enhancement of base pairing. And then, the idea that the translation initiation region (TIR) is usually targeted by sRNA was confirmed in vivo by antisense RNA stabilizing skills. To do so, twenty three artificial ncRNAs containing antisense regions to target mRNA were designed with double stem-loop stabilized structure, to examine...
Lee, Young-Hoonresearcher이영훈
한국과학기술원 : 화학과,
Issue Date
566085/325007  / 020088088

학위논문(박사) - 한국과학기술원 : 화학과, 2013.8, [ vi, 82 p. ]


artificial small RNA; 발현조절 메커니즘; Hfq; 유전자 발현저해; RNA 구조; 인공 small RNA; RNA scaffold; gene silencing; hfq; regulation mechanism

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