Riboswitches are regulatory elements of bacterial mRNA which function with conformational switching upon binding of specific cellular metabolites. In particular, transcriptional riboswitches regulate gene expression kinetically through the conformational change of the aptamer domain. In this study, we investigate the conformational dynamics and ligand binding mechanisms of the aptamer domain of a transcriptional prequeuosine (preQ) riboswitch from using two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) with microsecond time resolution. The obtained time-resolved single-molecule data indicate that the aptamer domain undergoes folding/unfolding including three forms, which are attributed to hairpin (O), pseudoknot-like (pF), and H-type pseudoknot (fF) structures. It is found that a cofactor, Mg, binds only to the fF form with the conformational selection mechanism. In contrast, it is indicated that the ligand, preQ, binds to the O form with the induced-fit mechanism and significantly accelerates the microsecond O → pF folding process. It is also shown that the binding with preQ substantially stabilizes the fF form that is generated from the pF form with a long time constant (>10 ms). Combining these results with the results of a former smFRET study on the slower time scale, we obtain an overall picture of the folding/unfolding dynamics of the aptamer domain as well as its energy landscape. On the basis of the picture obtained, we discuss the significance of the microsecond folding/unfolding of the aptamer domain for biological function of the riboswitch and propose the molecular mechanism of the gene expression controlled by the structural dynamics of the aptamer domain.
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