Characterization of Five Purine Riboswitches in Cellular and Cell-Free Expression Systems

Abstract

Bacillus subtilis employs five purine riboswitches for the control of purine de novo synthesis and transport at the transcription level. All of them are formed by a structurally conserved aptamer, and a variable expression platform harboring a rho-independent transcription terminator. In this study, we characterized all five purine riboswitches under the context of active gene expression processes both in vitro and in vivo. We identified transcription pause sites located in the expression platform upstream of the terminator of each riboswitch. Moreover, we defined a correlation between in vitro transcription readthrough and in vivo gene expression. Our in vitro assay demonstrated that the riboswitches operate in the micromolar range of concentration for the cognate metabolite. Our in vivo assay showed the dynamics of control of gene expression by each riboswitch. This study deepens the knowledge of the regulatory mechanism of purine riboswitches.

A journey into the regulatory secrets of the de novo purine nucleotide biosynthesis

“…In the case of PurR, (p)ppGpp compete for PRPP and thus promote PurR binding to DNA and repression of the transcription (Anderson et al, 2022). In B. subtilis, the single pur operon consisting of twelve DNPNB genes (see Figure 4B) is also controlled by a purine riboswitch, located between the promoter and the translation start site of the first gene, i.e., purE (Ebbole and Zalkin, 1987;Mandal et al, 2003;Lins et al, 2022). The purE riboswitch turns off gene expression when bound to guanine.…”