Fig. 3: [F30-Bro(+)]UTR(+) transcription by MS2 replicase in the absence and presence of various PURE enzymes. | Communications Biology

Fig. 3: [F30-Bro(+)]UTR(+) transcription by MS2 replicase in the absence and presence of various PURE enzymes.

From: In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity

Fig. 3

a DFHBI-1T fluorescence increase recorded after sample incubation at 37 °C for 1 h. Reactions contained 0.3 µM MS2rep, 50 nM [F30-Bro(−)]UTR(−), EF-Tu (15 µM), S1 (1.5 µM) and full LD2 (5 µM) except for the proteins indicated. Negative control (NC, dark grey) contained no MS2rep while the positive control (PC, blue) contained the full set of LD2 proteins and 0.3 µM MS2rep. b DFHBI-1T fluorescence as in a but in the presence of MS2rep and individual LD2 proteins alone (5 µM). The positive control (PC, blue) contained 0.3 µM MS2rep, 50 nM [F30-Bro(−)]UTR(−), EF-Tu (15 µM), EF-Ts (15 µM) and S1 (1.5 µM), and the negative control (NC) was identical with the exception of no MS2rep. c Co-factor titration assay. Reactions were carried out as in b but with varying concentrations of selected PURE proteins, or PEG 8000 to analyse excluded volume effects. Replication mixtures were programmed with 50 nM [F30-Bro(−)]UTR(−). Fluorescence was measured every minute over a 60-minute time course at 37 °C. Error bars indicate standard deviation based on three independent replicates prepared using the same protein stock solutions. The negative values for NC in panel a resulted from a baseline drift during the experiment.

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