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Reengineering a natural riboswitch by dual genetic selection.

Thiamine pyrophosphate (TPP) riboswitches are found in bacterial and eukaryotic mRNAs and regulate gene expression by switching the local RNA structure upon binding TPP to the natural RNA aptamer. We reengineered a natural TPP riboswitch which represses the downstream gene expression in response to TPP so that the downstream gene is activated in response to the ligand by dual genetic selection. We identified and characterized a number of artificial riboswitches which exhibit opposite response to TPP compared to the wild-type. The results suggest a remarkable flexibility of the riboswitch mechanism to implement different modes of gene regulation which may have been a beneficial feature for the primitive cells.