Computational protocol: Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

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Protocol publication

[…] To predict the secondary structure of the thiC riboswitch in the absence of TPP, intergenic sequences located upstream of the thiC were retrieved in proteobacteria using the RiboGap database (http://ribogap.iaf.inrs.ca). Using these sequences, RNA secondary structure prediction was performed using both LocaRNA and CMfinder from which a potential candidate exhibiting an unpaired RBS region was obtained. The validity of the prediction was further tested by analysing the stability of local optimal structures using RNAConSLOpt. Most predictions corresponded to OFF models, but the single predicted ON conformation was close to the LocaRNA and CMfinder models. The predicted secondary structure model for an alignment of 77 gammaproteobacteria () is represented in and shows the presence of an alternative pairing (anti-P1 stem) preventing both P1 and sequestrator helices (), consistent with a TPP-free ON state structure allowing efficient translation initiation. Using the ON state structure, an additional homology search was performed using INFERNAL on all intergenic regions with predicted TPP riboswitches in sequenced genomes and resulting hits were realigned with cmalign to confirm the presence of the model. A total of 127 additional sequences were found to contain the anti-P1 stem domain, but were not included due to additive minor divergences of the ON state riboswitch structure. In our model, the ON state structure of the thiC riboswitch () is mutually exclusive to the TPP-bound conformer, thereby providing a molecular mechanism explaining how the thiC riboswitch modulates translation initiation on TPP binding. […]

Pipeline specifications

Software tools LocARNA, CMfinder
Application RNA structure analysis
Organisms Escherichia coli, Bacteria