Development and Application of a Highly Efficient Protein Synthesis Technique Using Riboswitches in Microorganisms

Abstract

Riboswitches are conserved RNA elements that precisely and specifically target small molecules to control gene expression. For example, a thiamine pyrophosphate (TPP)-dependent riboswitch downregulates gene expression upon binding TPP in Aspergillus oryzae. Here we found that physiological concentrations of Mg2+ play a critical role in the regulation of TPP binding by the TPP-dependent riboswitch in A. oryzae. We further showed that the wild-type TPP-dependent riboswitch located in a 5′-untranslated region (UTR) intron of the thiA gene directly interacted with TPP and led to improper splicing, resulting in the retention of a remnant of the intron. Based on this mechanism, we constructed an “on riboswitch,” in which the remnant sequence inhibiting the gene expression was truncated from the intron, leading to proper splicing on TPP binding. The riboswitch developed here should be a useful tool for gene regulation.