Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Nucleic Acids Res. 2018 Sep 28;46(17):9094-9105. doi: 10.1093/nar/gky539.
Riboswitches are structured mRNA sequences that regulate gene expression by directly binding intracellular metabolites. Generating the appropriate regulatory response requires the RNA rapidly and stably acquire higher-order structure to form the binding pocket, bind the appropriate effector molecule and undergo a structural transition to inform the expression machinery. These requirements place riboswitches under strong kinetic constraints, likely restricting the sequence space accessible by recurrent structural modules such as the kink turn and the T-loop. Class-II cobalamin riboswitches contain two T-loop modules: one directing global folding of the RNA and another buttressing the ligand binding pocket. While the T-loop module directing folding is highly conserved, the T-loop associated with binding is substantially less so, with no clear consensus sequence. To further understand the functional role of the binding-associated module, a functional genetic screen of a library of riboswitches with the T-loop and its interacting nucleotides was used to build an experimental phylogeny comprised of sequences that possess a wide range of cobalamin-dependent regulatory activity. Our results reveal conservation patterns of the T-loop and its interaction with the binding core that allow for rapid tertiary structure formation and demonstrate its importance for generating strong ligand-dependent repression of mRNA expression.
Riboswitches 是一类结构型的 mRNA 序列，可通过直接结合细胞内代谢物来调节基因表达。为了产生适当的调控反应，RNA 需要快速且稳定地获取更高阶结构，形成结合口袋，结合适当的效应分子，并发生结构转变，从而通知表达机制。这些要求使 riboswitches 受到强烈的动力学限制，可能限制了诸如扭曲环和 T 环等反复出现的结构模块可访问的序列空间。类 II 钴胺素 riboswitches 包含两个 T 环模块：一个指导 RNA 的整体折叠，另一个支撑配体结合口袋。虽然指导折叠的 T 环模块高度保守，但与结合相关的 T 环则不然，没有明确的共识序列。为了进一步了解结合相关模块的功能作用，对带有 T 环及其相互作用核苷酸的 riboswitches 文库进行了功能遗传筛选，以构建一个实验系统发育树，该树由具有广泛钴胺素依赖性调控活性的序列组成。我们的结果揭示了 T 环及其与结合核心相互作用的保守模式，这些模式允许快速形成三级结构，并证明了其在生成强配体依赖性 mRNA 表达抑制中的重要性。
