Mutational analysis of structural elements in a class-I cyclic di-GMP riboswitch to elucidate its regulatory mechanism

Abstract

The Vc2 riboswitch possesses an aptamer domain belonging to the class-I c-di-GMP riboswitch family. This domain has been analysed and the molecular mechanism by which it recognizes the c-di-GMP ligand has been elucidated. On the other hand, the regulatory mechanism of the full-length Vc2 riboswitch to control its downstream open reading frame (ORF) remains largely unknown. In this study, we performed in vivo reporter assays and in vitro biochemical analyses of the full-length riboswitch and its aptamer domain. We evaluated the results of in vivo and in vitro analyses to elucidate the regulatory mechanism of the Vc2 riboswitch. The present results suggest that recognition of c-di-GMP ligand by the Vc2 riboswitch aptamer domain downregulates expression of its downstream ORF primarily at the translational level.

Recognition of cyclic‐di‐GMP by a riboswitch conducts translational repression through masking the ribosome‐binding site distant from the aptamer domain

“…The neighboring A91 was also fixed inside the riboswitch core upon c‐di‐GMP recognition (Kulshina et al., ; Smith et al., ). Ligand‐dependent enhancement of the protection was also observed at the P2‐GAAA loop and P3‐R(Vc2) motif, which also suggested that binding of c‐di‐GMP stabilizes the folded structure of the riboswitch, in which the P2‐GAAA loop is docked stably to the P3‐R(vc2) motif through tetraloop–receptor interaction (Inuzuka et al., ). Changes in the protection patterns at the aptamer domain strongly suggest that the recognition of c‐di‐GMP induces the ligand‐bound structure showed by X‐ray crystallographic studies.…”

“…The main objective of this study was to experimentally elucidate the ligand‐bound and ligand‐free structures of the riboswitch. However, a previous study involving chemical probing of the full‐length riboswitch RNA with dimethyl sulfate (DMS) showed no significant changes in modification pattern with/without c‐di‐GMP (Inuzuka et al., ). These results were due to the intrinsic flexibility of the riboswitch structures and also to the experimental conditions that may not have been optimized for DMS modifications.…”

“…We next examined DMS modification of 5′ UTR elements of the two mutant riboswitch under conditions of cotranscription with phage T7 RNA polymerase. Previous DMS modification successfully demonstrated the ligand‐bound form of the aptamer domain in the ΔA95 mutant riboswitch, whose P1 stem is stabilized by the deletion of A95 and preorganized in the absence of c‐di‐GMP (Inuzuka et al., ). The in vivo reporter assay and S30 assay showed that ΔA95 mutant has translation property similar to that of the P1‐ON mutant (Inuzuka et al., ).…”

“…Based on the structural probing of two mutants, we next analyzed the structures of the full‐length riboswitch in ligand‐bound and ligand‐free states. Cotranscription with phage T7 RNA polymerase, however, may not be suitable for examining folding of the wild‐type riboswitch (Inuzuka et al., ). Therefore, we examined synthesis of the wild‐type riboswitch RNA with E. coli RNA polymerase because this riboswitch has been shown to function under cell‐free transcription/translation conditions using E. coli S30 extract (Inuzuka et al., ; Watters, Strobel, Yu, Lis, & Lucks, ).…”

Ecological implications of gene regulation by TfoX and TfoY among diverse Vibrio species

“…In a previous study on V. cholerae , we showed that lowered c‐di‐GMP levels led to the production of TfoY under standard laboratory conditions (e.g. in LB medium) and we and others suggested that this c‐di‐GMP‐dependent control occurred at the translational level (Inuzuka et al ., ; Metzger et al ., ). Interestingly, Gode‐Potratz et al .…”