RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase.
Researchers at SUNY Buffalo have developed a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). The researchers demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, they show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. These findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.
Overview of TECprobe-VL
Randomly biotinylated DNA templates are in vitro transcribed to generate cotranscriptionally folded intermediate transcripts displayed from E. coli RNAP. The cotranscriptionally displayed intermediate transcripts are chemically probed and purified. An adapter is ligated to the RNA 3’ end and used to prime solid-phase error-prone reverse transcription. Full-length cDNA is amplified using primers that anneal to the adapter and the 5’ structure cassette. Following high-throughput sequencing, data are processed using fastp, TECtools, and ShapeMapper2 either with or without neighboring transcript smoothing. SC1 structure cassette 1, RT reverse transcription, ET SSB extreme thermostable single-stranded DNA binding protein.
Availability – The script used for RNA structure prediction can be accessed at https://github.com/e-strobel-lab/Publications.