TBI-p-2014-16

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Titel:
Engineering synthetic riboswitches acting on transcription

Author(s):
Wachsmuth M, Lorenz R, Serfling R, Findeiß S, Stadler PF and Mörl M

submitted to:
NAR

Abstract:
Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. This regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show how to change the terminator composition to enhance the regulatory potential of a transcriptional riboswitch responding to the binding of its specific ligand. Furthermore, in silico analyses predict terminator folding traps in nonfunctional constructs, impeding proper riboswitch-mediated regulation of transcription. A combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

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