Monitoring structural dynamics of nascent bacterial riboswitches responding to TPP within transcriptional complexes.

This chapter describes a single-molecule fluorescence resonance energy transfer (smFRET) approach to monitor cotranscriptional RNA folding within native transcription complexes. The approach combines stepwise transcription by the Escherichia coli RNA polymerase (RNAP) with site-specific RNA labeling with a Cy3-Cy5 FRET pair via copper-free click chemistry. The chapter also details single molecule approaches to monitor the structural aspects of nascent transcripts and the analysis of observed RNA populations. Using the thiamin pyrophosphate (TPP)-sensing E. coli tbpA riboswitch as a model system, we detail procedures for DNA template preparation, RNA labeling and smFRET acquisition. Key advantages of the described smFRET approach include the use of native transcripts and the ability to monitor RNA structural changes within transcription complexes that are either stalled at naturally occurring pause sites or that are actively elongating along the DNA template. This method permits the study of cotranscriptional regulatory mechanisms taking place either in bacterial or eukaryotic elongation complexes and could be extrapolated to the use of any biochemical probes compatible with the click chemistry approach.