ZTP riboswitch PK4 mutant cotranscriptional folding with 1 mM ZMP
Randomly biotinylated DNA templates were prepared that consisted of an E. coli promoter followed by DNA encoding the C. beijerinckii pfl Riboswitch PK4 mutant. DNA template was incubated with streptavidin after creating open complexes with E. coli RNA polymerase, followed by initiating in vitro transcription with the addition of Mg++. Reactions were run for 30s at 37C in standard transcription buffer. Reactions were then probed with BzCN, or a control solvent (DMSO), prior to extraction with Trizol. Extracted RNAs were then processed according to the SHAPE-Seq v2.1 protocol for cDNA synthesis and library preparation prior to sequencing (doi:10.1016/j.ymeth.2016.04.002).