Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan.
Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), 2-1 Hirosawa, Wako 351-0198, Japan.
J Am Chem Soc. 2021 Jun 2;143(21):7968-7978. doi: 10.1021/jacs.1c01077. Epub 2021 May 20.
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.
核糖开关是细菌 mRNA 的调控元件，通过结合特定的细胞代谢物，在构象转换中发挥作用。特别是转录核糖开关通过适体结构域的构象变化，在动力学上调节基因表达。在这项研究中，我们使用二维荧光寿命相关光谱（2D FLCS）以微秒时间分辨率，研究了转录前 Que 核苷酸（preQ）核糖开关的适体结构域的构象动力学和配体结合机制。所获得的时间分辨单分子数据表明，适体结构域经历了包括三种形式的折叠/展开，这归因于发夹（O）、假结样（pF）和 H 型假结（fF）结构。发现辅因子 Mg 仅以构象选择机制与 fF 形式结合。相比之下，表明配体 preQ 以诱导契合机制与 O 形式结合，并显著加速微秒 O→pF 折叠过程。还表明，与 preQ 的结合显著稳定了 fF 形式，该形式是由 pF 形式以长时间常数（>10ms）生成的。将这些结果与之前在较慢时间尺度上进行的 smFRET 研究结果结合起来，我们获得了适体结构域折叠/展开动力学及其能量景观的整体图像。基于获得的图像，我们讨论了适体结构域的微秒折叠/展开对核糖开关生物学功能的意义，并提出了由适体结构域的结构动力学控制的基因表达的分子机制。
