Different Folding Kinetics of Two Homologous Riboswitch Aptamer Domains.

Riboswitches are structured, modular non coding elements found essentially in bacterial mRNA (5'UTR) where they control the expression of the downstream gene at the transcriptional or translational level [1]. Few examples of riboswitches are known in eukaryotes where they control gene splicing or mRNA stability. Their aptamer domain binds small molecules (nucleotides, vitamins, ions...) with high specificity. This binding triggers the remodeling of the downstream expression platform, allowing the regulation process to occur. Riboswitches which bind thiamine pyrophosphate (TPP) form the largest family presently known across the three kingdoms of life.Using a time-resolved probing strategy [2], we have determined the folding kinetics of two homologous TPP binding riboswitch aptamer domains from E.coli and the plant A. thaliana. The E.coli riboswitch is located in the 5' UTR of the thiC gene and regulates gene expression at the transcriptional level while its plant homolog lies in the 3'UTR region of the thiC gene and regulates alternative splicing. At variance with the usual method yielding kinetic information residue by residue, all experimental data could be fit at once with a global kinetic model, which yielded the kOn for ligand binding and the folding kinetic constant kF. The results show that the E coli riboswitch folds faster than that of A.thaliana (typically, kF ~ 0.2 s-1 vs. 0.01 s-1) and that its folding rate is almost temperature independent, whereas kF for A.thaliana increases ca. 10-fold for every +10 °C variation. This suggests that the A.thaliana riboswitch may be viewed as a thermosensor.