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The cotranscriptional folding landscape for two cyclic di-nucleotide-sensing riboswitches with highly homologous aptamer domains acting either as ON- or OFF-switches.

ABSTRACT: Riboswitches are gene regulatory elements located in untranslated mRNA regions. They bind inducer molecules with high affinity and specificity. Cyclic-di-nucleotide-sensing riboswitches are major regulators of genes for the environment, membranes and motility (GEMM) of bacteria. Up to now, structural probing assays or crystal structures have provided insight into the interaction between cyclic-di-nucleotides and their corresponding riboswitches. ITC analysis, NMR analysis and computational modeling allowed us to gain a detailed understanding of the gene regulation mechanisms for the Cd1 (Clostridium difficile) and for the pilM (Geobacter metallireducens) riboswitches and their respective di-nucleotides c-di-GMP and c-GAMP. Binding capability showed a 25 nucleotide (nt) long window for pilM and a 61 nt window for Cd1. Within this window, binding affinities ranged from 35 μM to 0.25 μM spanning two orders of magnitude for Cd1 and pilM showing a strong dependence on competing riboswitch folds. Experimental results were incorporated into a Markov simulation to further our understanding of the transcriptional folding pathways of riboswitches. Our model showed the ability to predict riboswitch gene regulation and its dependence on transcription speed, pausing and ligand concentration.

SUBMITTER: Landgraf T

PROVIDER: S-EPMC9262584 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Adenine protonation enables cyclic-di-GMP binding to cyclic-GAMP sensing riboswitches.

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Design criteria for synthetic riboswitches acting on transcription.

Project description:Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

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Cotranscriptional folding kinetics of ribonucleic acid secondary structures.

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