Peltier Johann, Soutourina Olga
Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris Cedex 15, France.
Université Paris Diderot, Sorbonne Paris Cité, 25 rue du Dr Roux, 75724, Paris Cedex 15, France.
Methods Mol Biol. 2017;1657:377-402. doi: 10.1007/978-1-4939-7240-1_29.
Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important signaling molecule for community behavior control, cell morphogenesis, and virulence in bacteria. In addition to protein effectors, this second messenger binds RNA molecules that act as riboswitches to control target gene expression. In this chapter, we describe a method for experimental validation of the functionality of c-di-GMP-responsive riboswitches and the analysis of c-di-GMP control of target gene expression by qRT-PCR and Northern blot. This procedure can be used for the studies of in silico-predicted riboswitch candidates, as well as a targeted experimental approach for exploring the data from next-generation sequencing. The examples on the analysis of type I and type II c-di-GMP-responsive riboswitches in Clostridium difficile are provided to illustrate the application of the method.
双（3'-5'）-环二鸟苷单磷酸（c-di-GMP）是细菌中控制群体行为、细胞形态发生和毒力的重要信号分子。除了蛋白质效应器外，这种第二信使还与作为核糖开关的RNA分子结合，以控制靶基因表达。在本章中，我们描述了一种用于实验验证c-di-GMP响应核糖开关功能以及通过qRT-PCR和Northern印迹分析c-di-GMP对靶基因表达控制的方法。该程序可用于对计算机预测的核糖开关候选物的研究，以及用于探索来自下一代测序数据的靶向实验方法。提供了艰难梭菌中I型和II型c-di-GMP响应核糖开关分析的实例，以说明该方法的应用。
