Synthetic dual-function RNA reveals features necessary for target regulation

Abstract

Small base pairing RNAs (sRNAs) and small proteins comprise two classes of regulators that allow bacterial cells to adapt to a wide variety of growth conditions. A limited number of transcripts encoding both of these activities, regulation of mRNA expression by base pairing and a small regulatory protein, have been identified. Given that few have been characterized, little is known about the interplay between the two regulatory functions. To investigate the competition between the two activities, we constructed synthetic dual-function RNAs, hereafter referred to as MgtSR or MgtRS, comprised of the Escherichia coli sRNA MgrR and the open reading frame encoding the small protein MgtS. MgrR is a 98 nt base pairing sRNA that negatively regulates eptB encoding phosphoethanolamine transferase. MgtS is a 31 aa small inner membrane protein that is required for the accumulation of MgtA, a magnesium (Mg2+) importer. Expression of the separate genes encoding MgrR and MgtS is normally induced in response to low Mg2+ by the PhoQP two-component system. By generating various versions of this synthetic dual-function RNA, we probed how the organization of components and the distance between the coding and base pairing sequences contribute to the proper function of both activities of a dual-function RNA. By understanding the features of natural and synthetic dual-function RNAs, future synthetic molecules can be constructed to maximize their regulatory impact. IMPORTANCEDual-function RNAs encode a small protein and also base pair with mRNAs to act as small, regulatory RNAs. Given that only a limited number of dual-function RNAs have been characterized, further study of these regulators is needed to increase understanding of their features. This study demonstrates that a functional synthetic dual-regulator can be constructed from separate components and used to study the functional organization of dual-function RNAs, with the goal of exploiting these regulators.