Identification of c-di-GMP-Responsive Riboswitches

Methods Mol Biol. 2017:1657:377-402. doi: 10.1007/978-1-4939-7240-1_29.

Abstract

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.

Keywords: Northern blot; Premature termination of transcription; Riboswitches; Target gene; c-di-GMP; qRT-PCR.

MeSH terms

  • Blotting, Northern / methods
  • Clostridioides difficile / genetics
  • Clostridioides difficile / metabolism
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / metabolism
  • Nucleic Acid Conformation
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • Riboswitch*
  • Second Messenger Systems
  • Transcription Termination, Genetic
  • Transcription, Genetic
  • Workflow

Substances

  • RNA, Bacterial
  • Riboswitch
  • bis(3',5')-cyclic diguanylic acid
  • Cyclic GMP