Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution
Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution
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DOI：
10.1073/pnas.1218062110
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发表时间：
2013-03-12
影响因子：
11.1
通讯作者：
Micura, Ronald
中科院分区：
文献类型：
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作者：
Haller, Andrea;Altman, Roger B.;Micura, Ronald
硫胺素焦磷酸（TPP）敏感的mRNA结构域是已知的最普遍的核糖开关。尽管深入研究，复杂的配体识别和伴随的折叠过程中的TPP核糖开关，最终在基因表达的调控仍然难以捉摸。在这里，我们使用单分子荧光共振能量转移成像来探测在镁和TPP存在和不存在的情况下TPP适体结构域的折叠景观。为此，使用不同的标记模式来感测适体结构域的开关螺旋（P1）和两个传感器臂（P2/P3和P4/P5）的动力学。后者的结构元件使域间三级接触（L5/P3），跨越紧邻配体结合位点的区域。在每种情况下，TPP核糖开关的构象动力学受配体结合的影响。由适体结构域的5 '和3'端形成的P1开关螺旋在单独的Mg 2+存在下采用主要折叠的结构。然而，即使在饱和浓度的Mg 2+和TPP，P1螺旋，以及周围的TPP结合位点的远端区域，表现出意想不到的程度的残留动力学和分散动力学行为。这种可塑性导致P3/P5前臂在开放和闭合配置之间持续交换，这可能有助于TPP配体的进入和退出。相应地，我们认为TPP适体结构域的这些特征直接有助于核糖开关介导的翻译调控机制。
Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligand-binding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5' and 3' ends of the aptamer domain, adopts a predominantly folded structure in the presence of Mg2+ alone. However, even at saturating concentrations of Mg2+ and TPP, the P1 helix, as well as distal regions surrounding the TPP-binding site, exhibit an unexpected degree of residual dynamics and disperse kinetic behaviors. Such plasticity results in a persistent exchange of the P3/P5 forearms between open and closed configurations that is likely to facilitate entry and exit of the TPP ligand. Correspondingly, we posit that such features of the TPP aptamer domain contribute directly to the mechanism of riboswitch-mediated translational regulation.