Department of Chemistry and Biochemistry, University of Colorado at Boulder, 596 UCB, Boulder, CO 80309-0596, USA.
J Mol Biol. 2012 Oct 12;423(1):17-33. doi: 10.1016/j.jmb.2012.06.038. Epub 2012 Jul 3.
A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, we have determined the relative contributions of the polar functional groups to binding affinity and the regulatory response. Our results reveal that the lysine riboswitch has >1000-fold specificity for lysine over other amino acids. The aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main-chain functional groups in order to achieve this specificity. At low nucleotide triphosphate (NTP) concentrations, we observe good agreement between the half-maximal regulatory activity (T(50)) and the affinity of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 μM [NTP], the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that, under physiologically relevant conditions, riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell.
在细菌中，调节基因表达的一种普遍方式是通过 mRNA 前导序列中的核糖开关。与蛋白质抑制剂一样，这些 RNA 元件必须与效应分子高度特异性结合，以针对具有相似化学结构的其他细胞代谢物的背景，从而引发适当的调节反应。当前的赖氨酸核糖开关晶体结构并不能完全理解选择性，因为识别主要通过氨基酸的主链原子介导。使用一组定向的赖氨酸类似物和其他氨基酸，我们确定了极性官能团对结合亲和力和调节反应的相对贡献。我们的结果表明，赖氨酸核糖开关对赖氨酸的特异性是其他氨基酸的 1000 倍以上。适体对 ε-氨基的精确位置高度敏感，并且对主链官能团的改变具有相对的耐受性，以实现这种特异性。在低核苷酸三磷酸 (NTP) 浓度下，我们观察到受体对赖氨酸的半最大调节活性 (T(50)) 和亲和力之间存在良好的一致性，以及许多类似物。然而，在 400 μM [NTP] 以上时，引发转录终止所需的赖氨酸浓度升高，使核糖开关进入动力学控制状态。这些数据表明，在生理相关条件下，核糖开关可以整合效应物和 NTP 浓度，以产生适合细胞整体代谢状态的调节反应。
