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

Abstract

The riboswitch is a class of RNA-based gene regulatory machinery that is dependent on recognition of its target ligand by RNA tertiary structures. Ligand recognition is achieved by the aptamer domain, and ligand-dependent structural changes of the expression platform then usually mediate termination of transcription or translational initiation. Ligand-dependent structural changes of the aptamer domain and expression platform have been reported for several riboswitches with short (<40 nucleotides) expression platforms. In this study, we characterized structural changes of the Vc2 c-di-GMP riboswitch that represses translation of downstream open reading frames in a ligand-dependent manner. The Vc2 riboswitch has a long (97 nucleotides) expression platform, but its structure and function are largely unknown. Through mutational analysis and chemical probing, we identified its secondary structures that are possibly responsible for switch-OFF and switch-ON states of translational initiation.

c-di-GMP inhibits LonA-dependent proteolysis of TfoY in Vibrio cholerae

“…Levels of c-di-GMP are elevated in biofilms, thus it is possible that TfoY may aid in dispersal from biofilms by enhancing motility. Conversely, we did not observe TfoY to significantly influence motility when c-di-GMP levels were low, which is consistent with a prior analysis that analyzed TfoY-dependent [24,26,28]. In contrast, transcripts produced from promoters P3 and P4 (red bent arrows), driven by the c-di-GMP binding transcriptional activator VpsR, do not contain the Vc2 riboswitch [26].…”

“…Notably, levels of TfoY were significantly enriched in the low c-di-GMP strain in the absence of lonA . It was previously shown that c-di-GMP limits production of TfoY protein by binding to a riboswitch (Vc2) located in the 5’UTR of tfoY mRNA [ 24 , 27 , 28 ]. Thus, this result is consistent with prior analyses, which have shown that decreasing levels of c-di-GMP results in enhanced TfoY translation, and suggests that LonA plays a more significant role in regulating levels of TfoY when c-di-GMP levels are low [ 24 , 26 ].…”

“…Promoters P1 and P2 produce transcript that contains the Vc2 riboswitch, which restricts translation when bound by c-di-GMP [ 24 , 27 , 28 ]. Decreasing levels of c-di-GMP derepresses the Vc2 inhibitory mechanism and permits translation of tfoY mRNA into TfoY protein [ 24 , 26 – 28 ]. However, elevated levels of c-di-GMP also permit the production of TfoY [ 26 ].…”

“…The upstream regulatory region of tfoY contains 4 promoters. Promoters P1 and P2 (black bent arrows) produce transcript that contains the Vc2 riboswitch (close stem loop), which functions as an off switch when bound by c-di-GMP (yellow circle) and prevents translation [ 24 , 26 , 28 ]. In contrast, transcripts produced from promoters P3 and P4 (red bent arrows), driven by the c-di-GMP binding transcriptional activator VpsR, do not contain the Vc2 riboswitch [ 26 ].…”

“…To date, only two known LonA substrates have been identified in V. cholerae. The first is FliA, an alternative sigma factor (σ 28 ) that coordinates the activation of late stage flagellar genes and the repression of virulence gene expression [8]. The second is the quorum sensing master regulator HapR, which is proteolyzed by LonA upon heat shock in order to induce biofilm formation [23].…”