Mayer Günter, Famulok Michael
Rheinische Friedrich-Wilhelms-Universität Bonn Life and Medical Sciences Institute, Gerhard-Domagk-Str.1, 53121, Bonn, Germany.
Methods Mol Biol. 2009;540:291-300. doi: 10.1007/978-1-59745-558-9_21.
Riboswitches are non-coding RNA elements mainly located in the 5' untranslated regions (UTR) of bacterial genes. They bind to small metabolites and upon binding conformational changes occur that trigger the expression of a certain gene. Riboswitches have been identified that bind to amino acids, purines, and other small metabolites such as thiamine pyrophosphate. Riboswitches contain an aptamer domain which is necessary for interaction with the metabolite and a related expression domain which harbours structural and sequence information required for interference with gene expression. The binding of a metabolite to the aptamer domain induces structural rearrangements that are relayed to the expression domain, thereby interfering with gene expression. To investigate and determine domains of the riboswitches which undergo conformational changes upon metabolite binding we used a dynamic SELEX process and identified RNA aptamers that bind to the metabolite-free variant of the riboswitch but are released upon metabolite addition. By this means, and after determination of the binding region, domains which are necessary for proper function of a full-length riboswitch can be identified.
核糖开关是非编码RNA元件,主要位于细菌基因的5'非翻译区(UTR)。它们与小分子代谢物结合,结合后会发生构象变化,从而触发特定基因的表达。已鉴定出能与氨基酸、嘌呤以及其他小分子代谢物(如硫胺素焦磷酸)结合的核糖开关。核糖开关包含一个适体结构域,这是与代谢物相互作用所必需的,以及一个相关的表达结构域,该结构域包含干扰基因表达所需的结构和序列信息。代谢物与适体结构域的结合会诱导结构重排,这种重排会传递到表达结构域,从而干扰基因表达。为了研究和确定核糖开关在与代谢物结合时发生构象变化的结构域,我们采用了动态SELEX过程,并鉴定出了与核糖开关的无代谢物变体结合但在添加代谢物后会释放的RNA适体。通过这种方法,在确定结合区域后,可以鉴定出全长核糖开关正常功能所必需的结构域。