Department of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, PO Box 9502, Leiden, The Netherlands.
ACS Chem Biol. 2013 Apr 19;8(4):733-40. doi: 10.1021/cb300629b. Epub 2013 Jan 25.
Knowing the molecular details of the interaction between riboswitch aptamers and their corresponding metabolites is important to understand gene expression. Here we report on a novel in vitro assay to study preQ(1) riboswitch aptamers upon binding of 7-aminomethyl-7-deazaguanine (preQ(1)). The assay is based on the ability of the preQ(1) aptamer to fold, upon ligand binding, into a pseudoknotted structure that is capable of stimulating -1 ribosomal frameshifting (-1 FS). Aptamers from three different species were found to induce between 7% and 20% of -1 FS in response to increasing preQ(1) levels, whereas preQ(1) analogues were 100-1000-fold less efficient. In depth mutational analysis of the Fusobacterium nucleatum aptamer recapitulates most of the structural details previously identified for preQ(1) aptamers from other bacteria by crystallography and/or NMR spectroscopy. In addition to providing insight into the role of individual nucleotides of the preQ(1) riboswitch aptamer in ligand binding, the presented system provides a valuable tool to screen small molecules against bacterial riboswitches in a eukaryotic background.
了解核糖开关适体与其相应代谢物之间相互作用的分子细节对于理解基因表达非常重要。在这里，我们报告了一种新的体外测定法，用于研究在结合 7-氨基甲基-7-脱氮鸟嘌呤（preQ（1））时的 preQ（1）核糖开关适体。该测定法基于 preQ（1）适体的能力，即在配体结合后折叠成具有 -1 核糖体移码（-1 FS）能力的假结结构。发现来自三个不同物种的适体在响应增加的 preQ（1）水平时诱导 -1 FS 发生 7%至 20%之间的变化，而 preQ（1）类似物的效率低 100-1000 倍。对梭菌属核梭杆菌适体的深入突变分析重现了先前通过晶体学和/或 NMR 光谱学鉴定的来自其他细菌的 preQ（1）适体的大多数结构细节。除了深入了解配体结合中 preQ（1）核糖开关适体单个核苷酸的作用外，所提出的系统还提供了一种在真核背景下筛选针对细菌核糖开关的小分子的有价值的工具。
