National Heart, Lung and Blood Institute, 50 South Drive, MSC 8012, Bethesda, Maryland 20892-8012, USA.
Nature. 2013 Aug 15;500(7462):363-6. doi: 10.1038/nature12440. Epub 2013 Jul 28.
In Gram-positive bacteria, T-box riboswitches regulate the expression of aminoacyl-tRNA synthetases and other proteins in response to fluctuating transfer RNA aminoacylation levels under various nutritional states. T-boxes reside in the 5'-untranslated regions of the messenger RNAs they regulate, and consist of two conserved domains. Stem I contains the specifier trinucleotide that base pairs with the anticodon of cognate tRNA. 3' to stem I is the antiterminator domain, which base pairs with the tRNA acceptor end and evaluates its aminoacylation state. Despite high phylogenetic conservation and widespread occurrence in pathogens, the structural basis of tRNA recognition by this riboswitch remains ill defined. Here we demonstrate that the ~100-nucleotide T-box stem I is necessary and sufficient for specific, high-affinity (dissociation constant (Kd) ~150 nM) tRNA binding, and report the structure of Oceanobacillus iheyensis glyQ stem I in complex with its cognate tRNA at 3.2 Å resolution. Stem I recognizes the overall architecture of tRNA in addition to its anticodon, something accomplished by large ribonucleoproteins such as the ribosome, or proteins such as aminoacyl-tRNA synthetases, but is unprecedented for a compact mRNA domain. The C-shaped stem I cradles the L-shaped tRNA, forming an extended (1,604 Å(2)) intermolecular interface. In addition to the specifier-anticodon interaction, two interdigitated T-loops near the apex of stem I stack on the tRNA elbow in a manner analogous to those of the J11/12-J12/11 motif of RNase P and the L1 stalk of the ribosomal E-site. Because these ribonucleoproteins and T-boxes are unrelated, this strategy to recognize a universal tRNA feature probably evolved convergently. Mutually induced fit of stem I and the tRNA exploiting the intrinsic flexibility of tRNA and its conserved post-transcriptional modifications results in high shape complementarity, which in addition to providing specificity and affinity, globally organizes the T-box to orchestrate tRNA-dependent transcription regulation.
在革兰氏阳性菌中，T 盒核糖体开关通过响应各种营养状态下转移 RNA 氨酰化水平的波动，调节氨酰-tRNA 合成酶和其他蛋白质的表达。T 盒位于它们调节的信使 RNA 的 5'非翻译区，由两个保守结构域组成。茎 I 包含与对应 tRNA 的反密码子配对的特异性三联体核苷酸。茎 I 的 3'端是终止子结构域，与 tRNA 受体末端配对，并评估其氨酰化状态。尽管具有高度的系统发育保守性并且在病原体中广泛存在，但这种核糖体开关识别 tRNA 的结构基础仍未得到明确定义。在这里，我们证明了100 个核苷酸的 T 盒茎 I 是特异性、高亲和力（解离常数（Kd）150 nM）tRNA 结合所必需且足够的，并报告了 3.2 Å 分辨率下海洋芽孢杆菌 glyQ 茎 I 与它的对应 tRNA 复合物的结构。茎 I 除了反密码子外，还识别 tRNA 的整体结构，这是核糖体等大型核糖核蛋白或氨酰-tRNA 合成酶等蛋白质完成的，但对于紧凑的 mRNA 结构域来说是前所未有的。C 形的茎 I 环抱 L 形 tRNA，形成一个扩展的（1604 Å(2)）分子间界面。除了特异性-反密码子相互作用外，茎 I 顶点附近的两个互穿插的 T 环以类似于 RNase P 的 J11/12-J12/11 基序和核糖体 E 位的 L1 茎的方式堆积在 tRNA 的肘上。由于这些核糖核蛋白和 T 盒没有关系，因此这种识别通用 tRNA 特征的策略可能是通过趋同进化而来的。茎 I 和 tRNA 的相互诱导适合利用 tRNA 的固有灵活性及其保守的转录后修饰，导致高度的形状互补性，除了提供特异性和亲和力外，还全局组织 T 盒以协调 tRNA 依赖的转录调节。
