Dataset Information

What defines a synthetic riboswitch? - Conformational dynamics of ciprofloxacin aptamers with similar binding affinities but varying regulatory potentials.

ABSTRACT: Among the many in vitro-selected aptamers derived from SELEX protocols, only a small fraction has the potential to be applied for synthetic riboswitch engineering. Here, we present a comparative study of the binding properties of three different aptamers that bind to ciprofloxacin with similar KD values, yet only two of them can be applied as riboswitches. We used the inherent ligand fluorescence that is quenched upon binding as the reporter signal in fluorescence titration and in time-resolved stopped-flow experiments. Thus, we were able to demonstrate differences in the binding kinetics of regulating and non-regulating aptamers. All aptamers studied underwent a two-step binding mechanism that suggests an initial association step followed by a reorganization of the aptamer to accommodate the ligand. We show that increasing regulatory potential is correlated with a decreasing back-reaction rate of the second binding step, thus resulting in a virtually irreversible last binding step of regulating aptamers. We suggest that a highly favoured structural adaption of the RNA to the ligand during the final binding step is essential for turning an aptamer into a riboswitch. In addition, our results provide an explanation for the fact that so few aptamers with regulating capacity have been found to date. Based on our data, we propose an adjustment of the selection protocol for efficient riboswitch detection.

SUBMITTER: Kaiser C

PROVIDER: S-EPMC8053125 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Expression of a large fraction of genes in bacteria is controlled by riboswitches, which are found in the untranslated region of mRNA. Structurally riboswitches have a conserved aptamer domain to which a metabolite binds, resulting in a conformational change in the downstream expression platform. Prediction of the functions of riboswitches requires a quantitative description of the folding landscape so that the barriers and time scales for the conformational change in the switching region in the aptamer can be estimated. Using a combination of all atom molecular dynamics (MD) and coarse-grained model simulations we studied the response of adenine (A) binding add and pbuE A-riboswitches to mechanical force. The two riboswitches contain a structurally similar three-way junction formed by three paired helices, P1, P2, and P3, but carry out different functions. Using pulling simulations, with structures generated in MD simulations, we show that after P1 rips the dominant unfolding pathway in the add A-riboswitch is the rupture of P2 followed by unraveling of P3. In the pbuE A-riboswitch, after P1 unfolds P3 ruptures ahead of P2. The order of unfolding of the helices, which is in accord with single molecule pulling experiments, is determined by the relative stabilities of the individual helices. Our results show that the stability of isolated helices determines the order of assembly and response to force in these non-coding regions. We use the simulated free energy profile for the pbuE A-riboswitch to estimate the time scale for allosteric switching, which shows that this riboswitch is under kinetic control lending additional support to the conclusion based on single molecule pulling experiments. A consequence of the stability hypothesis is that a single point mutation (U28C) in the P2 helix of the add A-riboswitch, which increases the stability of P2, would make the folding landscapes of the two riboswitches similar. This prediction can be tested in single molecule pulling experiments.

Project description:In comparison to protein coding sequences, the impact of mutation and natural selection on the sequence and function of non-coding (ncRNA) genes is not well understood. Many ncRNA genes are narrowly distributed to only a few organisms, and appear to be rapidly evolving. Compared to protein coding sequences, there are many challenges associated with assessment of ncRNAs that are not well addressed by conventional phylogenetic approaches, including: short sequence length, lack of primary sequence conservation, and the importance of secondary structure for biological function. Riboswitches are structured ncRNAs that directly interact with small molecules to regulate gene expression in bacteria. They typically consist of a ligand-binding domain (aptamer) whose folding changes drive changes in gene expression. The glycine riboswitch is among the most well-studied due to the widespread occurrence of a tandem aptamer arrangement (tandem), wherein two homologous aptamers interact with glycine and each other to regulate gene expression. However, a significant proportion of glycine riboswitches are comprised of single aptamers (singleton). Here we use graph clustering to circumvent the limitations of traditional phylogenetic analysis when studying the relationship between the tandem and singleton glycine aptamers. Graph clustering enables a broader range of pairwise comparison measures to be used to assess aptamer similarity. Using this approach, we show that one aptamer of the tandem glycine riboswitch pair is typically much more highly conserved, and that which aptamer is conserved depends on the regulated gene. Furthermore, our analysis also reveals that singleton aptamers are more similar to either the first or second tandem aptamer, again based on the regulated gene. Taken together, our findings suggest that tandem glycine riboswitches degrade into functional singletons, with the regulated gene(s) dictating which glycine-binding aptamer is conserved.

Dataset Information

What defines a synthetic riboswitch? - Conformational dynamics of ciprofloxacin aptamers with similar binding affinities but varying regulatory potentials.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets