Kinetic Variability at the 5’ UTR of Bacterial Mrna Leads to Narrow Dynamic Range and High Sensitivity in 30S Ribosome Binding

The 7-aminomethyl-7-deazaguanine (preQ1)-sensing translational riboswitch in Thermoanaerobacter tengcongensis (Tte) is a dynamic RNA motif, embedded in the 5’- untranslated region of its mRNA. It regulates translation initiation in response to preQ1 by changing its conformation and partially sequestering the Shine-Dalgarno (SD) sequence from binding 30S ribosomal subunit. However, the kinetics of such ligand-dependent recognition of the SD sequence by a 30S subunit is poorly understood. Integrating single-molecule fluorescence microscopy with classical binding and translation initiation assays, we investigated the ligand-dependent accessibility of the SD sequence within the riboswitch. We observed long and short binding interactions of the 30S ribosomal subunit. The long events represent “initiation-active” binding, where the 30S subunit finds the desired SD region for initiating translation. In contrast, the short events represent transient, “exploratory”, binding. Increasing ligand concentration promotes the ON rate (kON) of 30S binding, consistent with the partial sequestration of the SD sequence due to ligand binding that impedes the access of the 30S ribosome to the SD sequence. Mutational studies of the mRNA further revealed enhanced binding of the 30S subunit when the SD sequence was moved downstream or increased in length. Our results support a model wherein translational riboswitches with weak sequestration of the SD sequence work as “modulator” rather than ON/OFF switches to control gene expression before the mRNA has finished transcription. While the effect on ribosome binding due to such partial sequestration goes undetected in bulk assays, our single-molecule assay detected such kinetic variability with high precision and sensitivity. Through this work, we established that ligand binding along with RNA structure and availability of the ribosome binding site play important roles in deciding the fate of translation in a thermophilic bacterium.