1 research outputs found
Retargeting a Dual-Acting sRNA for Multiple mRNA Transcript Regulation
Multitargeting small regulatory RNAs
(sRNAs) represent a potentially
useful tool for metabolic engineering applications. Natural multitargeting
sRNAs govern bacterial gene expression by binding to the translation
initiation regions of protein-coding mRNAs through base pairing. We
designed an <i>Escherichia coli</i> based genetic system
to create and assay dual-acting retargeted-sRNA variants. The variants
can be assayed for coordinate translational regulation of two alternate
mRNA leaders fused to independent reporter genes. Accordingly, we
began with the well-characterized <i>E. coli</i> native
DsrA sRNA. The merits of using DsrA include its well-characterized
separation of function into two independently folded stem-loop domains,
wherein alterations at one stem do not necessarily abolish activity
at the other stem. Expression of the sRNA and each reporter mRNA was
independently controlled by small inducer molecules, allowing precise
quantification of the regulatory effects of each sRNA:mRNA interaction <i>in vivo</i> with a microtiter plate assay. Using this system,
we semirationally designed DsrA variants screened in <i>E. coli</i> for their ability to regulate key mRNA leader sequences from the <i>Clostridium acetobutylicum n</i>-butanol synthesis pathway.
To coordinate intervention at two points in a metabolic pathway, we
created bifunctional sRNA prototypes by combining sequences from two
singly retargeted DsrA variants. This approach constitutes a platform
for designing sRNAs to specifically target arbitrary mRNA transcript
sequences, and thus provides a generalizable tool for retargeting
and characterizing multitarget sRNAs for metabolic engineering