All single-round in vitro transcription reactions for TECprobe-ML experiments were performed as 60 μl reactions containing 1X Transcription Buffer [20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol (DTT), and 0.1 mM EDTA], 0.1 mg/ml Molecular Biology-Grade BSA (Invitrogen), 100 μM high-purity NTPS (Cytiva), 10 nM randomly biotinylated template DNA, and 0.024 U/μl E. coli RNA polymerase holoenzyme (New England Biolabs). Single-round in vitro transcription reactions for TECprobe-SL experiments were performed as 60 μl reactions containing the same reagents as TECprobe-ML experiments except that the reactions used a 10 nM template DNA that contains an internal etheno-dA stall site downstream of a Cbe pfl ZTP riboswitch variant in which the poly-U tract was removed, and either 100 or 500 μM high-purity NTPs, as indicated. Transcription reactions for experiments where DMS was used to probe RNA structures contained additional Tris-HCl (pH 8.0) at final concentration of 100 mM to minimize pH changes during the chemical probing reaction. At the time of preparation, each TECprobe-ML reaction was 48 μl due to the omission of 10X (1 μM) streptavidin (Promega) and 10X Start Solution [100 mM MgCl2, 100 μg/ml rifampicin (Gold Biotechnology)] from the reaction. Each TECprobe-SL reaction was 54 μl due to the omission of 10X Start Solution from the reaction.
The composition of the single-round in vitro transcription master mix varied depending on the riboswitch system that was assessed. In vitro transcription reactions for the Cbe pfl ZTP riboswitch contained 2% (v/v) DMSO and, when present, 1 mM ZMP (Sigma-Aldrich). In vitro transcription reactions for the Bce crcB fluoride riboswitch that were performed in the presence of fluoride contained 10 mM NaF (Sigma-Aldrich). In vitro transcription reactions for the Cba ppGpp riboswitch and C69A ppGpp riboswitch variant contained 500 nM NusA and, when present, 250 μM ppGpp (Guanosine-3’,5’-bisdiphosphate) (Jena Bioscience).
Single-round in vitro transcription reactions were incubated at 37°C for 10 min to form open promoter complexes. For TECprobe-ML reactions, 6 μl of 1 μM streptavidin was then added for a final concentration of 100 nM streptavidin, and reactions were incubated for an additional 10 min at 37°C; TECprobe-SL reactions did not include streptavidin but were still incubated for a total of 20 min at 37°C. Transcription was initiated by adding 6 μl of 10X Start Solution to the reaction for a final concentration of 10 mM MgCl2 and 10 μg/ml rifampicin. The transcription reaction was incubated at 37°C for 2 min before chemical probing was performed as described below in the sectionRNA chemical probing .
The composition of the single-round in vitro transcription master mix varied depending on the riboswitch system that was assessed. In vitro transcription reactions for the Cbe pfl ZTP riboswitch contained 2% (v/v) DMSO and, when present, 1 mM ZMP (Sigma-Aldrich). In vitro transcription reactions for the Bce crcB fluoride riboswitch that were performed in the presence of fluoride contained 10 mM NaF (Sigma-Aldrich). In vitro transcription reactions for the Cba ppGpp riboswitch and C69A ppGpp riboswitch variant contained 500 nM NusA and, when present, 250 μM ppGpp (Guanosine-3’,5’-bisdiphosphate) (Jena Bioscience).
Single-round in vitro transcription reactions were incubated at 37°C for 10 min to form open promoter complexes. For TECprobe-ML reactions, 6 μl of 1 μM streptavidin was then added for a final concentration of 100 nM streptavidin, and reactions were incubated for an additional 10 min at 37°C; TECprobe-SL reactions did not include streptavidin but were still incubated for a total of 20 min at 37°C. Transcription was initiated by adding 6 μl of 10X Start Solution to the reaction for a final concentration of 10 mM MgCl2 and 10 μg/ml rifampicin. The transcription reaction was incubated at 37°C for 2 min before chemical probing was performed as described below in the section
