Construction of an RNA-expressing vector and its application to gene regulation in Escherichia coli

Abstract

Non-protein-coding RNAs (ncRNAs) employ a wide range of cellular functions from protein synthesis to regulation of gene expression. Mostly, ncRNAs, as inhibitors, repress the expression of target messenger RNA (mRNA) through formation of RNA-RNA duplex, such as antisense RNA. In contrast, a few ncRNAs stabilize target mRNAs and increase their protein expression, either directly or via effects on translation. In the recent study, the addition of increasing amounts of cognate antisense RNA decreases the cellular concentration of the target RNA in Escherichia coli. For more detailed control of ncRNA or antisense RNA with stable system, I constructed RNA expression plasmid vector by using the P1 stem of M1 RNA. The P1 stem is essential for M1 RNA stability, a catalytic unit of RNase P ribozyme. The prototypical vector was successfully constructed and antisense sequences were subcloned into the plasmid. The antisense RNAs were designed to be targeted to rnpB-CAT-lacZ fusion mRNA, and generally decreased β-galactosidase activity up to 32% in E. coli. Since it is possible that the structural difference or mismatching level leads to the different regulatory consequence for target RNAs, I set to generate the new regulatory RNA molecules from antisense RNA molecules. For this purpose, we constructed randomly 10% mutated antisense RNA libraries. I screened several clones from the libraries and found that they show large variance of β-galactosidase activity. These data imply that new types of regulatory RNA molecules can be generated from antisense RNA molecules by minor sequence changes.