Theoretical Biology and Biophysics Group, Theoretical Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Nucleic Acids Res. 2013 Feb 1;41(3):1922-35. doi: 10.1093/nar/gks978. Epub 2012 Dec 20.
Riboswitch operation involves the complex interplay between the aptamer domain and the expression platform. During transcription, these two domains compete against each other for shared sequence. In this study, we explore the cooperative effects of ligand binding and Magnesium interactions in the SAM-I riboswitch in the context of aptamer collapse and anti-terminator formation. Overall, our studies show the apo-aptamer acts as (i) a pre-organized aptamer competent to bind ligand and undergo structural collapse and (ii) a conformation that is more accessible to anti-terminator formation. We show that both Mg(2+) ions and SAM are required for a collapse transition to occur. We then use competition between the aptamer and expression platform for shared sequence to characterize the stability of the collapsed aptamer. We find that SAM and Mg(2+) interactions in the aptamer are highly cooperative in maintaining switch polarity (i.e. aptamer 'off-state' versus anti-terminator 'on-state'). We further show that the aptamer off-state is preferentially stabilized by Mg(2+) and similar divalent ions. Furthermore, the functional switching assay was used to select for phosphorothioate interference, and identifies potential magnesium chelation sites while characterizing their coordinated role with SAM in aptamer stabilization. In addition, we find that Mg(2+) interactions with the apo-aptamer are required for the full formation of the anti-terminator structure, and that higher concentrations of Mg(2+) (>4 mM) shift the equilibrium toward the anti-terminator on-state even in the presence of SAM.
核糖开关的运作涉及到适体结构域和表达平台之间的复杂相互作用。在转录过程中，这两个结构域会相互竞争，争夺共享序列。在这项研究中，我们在适体解链和反终止子形成的背景下，研究了配体结合和镁离子相互作用对 SAM-I 核糖开关的协同效应。总的来说，我们的研究表明，apo-aptamer 可以充当：(i) 一个有能力结合配体并发生结构解链的预先组织好的适体；(ii) 一种更容易形成反终止子的构象。我们表明，镁离子和 SAM 对于解链转变的发生都是必需的。然后，我们利用适体和表达平台之间共享序列的竞争来表征解链适体的稳定性。我们发现，SAM 和镁离子在适体中的相互作用在维持开关极性（即适体“关闭状态”与反终止子“开启状态”）方面具有高度的协同性。我们进一步表明，apo-aptamer 的关闭状态优先被镁离子和类似的二价离子稳定。此外，我们还使用功能切换测定法来选择硫代磷酸酯干扰，并确定潜在的镁螯合位点，同时描述它们与 SAM 一起在适体稳定化中的协调作用。此外，我们发现，apo-aptamer 与镁离子的相互作用对于完全形成反终止子结构是必需的，并且即使存在 SAM，较高浓度的镁离子（>4mM）也会使平衡向反终止子开启状态移动。
