SprA complexes were purified from a Δplug background (or from a ΔporV background for the control experiment in Extended Data Fig. 8b ) using the procedure described above and then transferred into buffer B (PBS pH 7.4 containing 0.005% LMNG) by dialysis. Of the SprA complexes, 300–400 pmol were mixed with the selected mCherry–CTD fusion at a 1:2–1:5 molar ratio in a final volume of 400 μl of buffer B and incubated overnight at 4 °C with constant agitation. The samples were then injected onto a Superose 6 Increase 10/300 GL size exclusion column (Cytiva) previously equilibrated in buffer B and the fluorescence (587 nm excitation, 610 nm emission) of the eluted material monitored using a Prominence fluorescence detector (RF-20AXS, Shimadzu). Samples for cryo-EM were crosslinked directly before the size exclusion step by addition of 3.5 μl of a 25% (w/v) glutaraldehyde solution. The samples were then incubated for 60 min on ice before the reaction was quenched by addition of 100 μl of buffer W. Peak fractions from the size exclusion column containing SprA–mCherry–CTD complexes were concentrated using a 100-kDa-MWCO Vivaspin 500 column.
Superose 6 increase 10 300 gl size exclusion column
Manufactured by Cytiva
Superose 6 Increase 10/300 GL is a size exclusion chromatography column used for the separation and purification of proteins, peptides, and other biomolecules based on their molecular size and shape. The column has a bed volume of 24 mL and is made with a stable, cross-linked agarose matrix that can withstand high flow rates and pressures.
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3 protocols using superose 6 increase 10 300 gl size exclusion column
1
Purification and Characterization of SprA Complexes
2
Reconstitution of NarL-RNAP Complex
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NarL-TAC was assembled as follows: Core RNAP (α2ββ'ω) was mixed with the annealed yeaR promoted DNA scaffold and free NTPs (GTP and ATP), then incubated at 37°C for 1 min. Obtained complex was mixed with σ70 and further incubated at 37°C for 1 min. Lastly, phosphorylated NarL was added to the mixture and incubated at 37°C for 8 minutes. Complex components were mixed at the final 1:2:2.4 molar ratio of RNAP holoenzyme/promoter DNA/Phosphorylated NarL respectively. Free NTPs for the de novo RNA synthesis were added to the final concentration of 0.2 mM. Assembly reaction was performed in the 500 μl volume, loaded onto Superose® 6 Increase 10/300 GL size exclusion column (Cytiva). Elution fractions were analyzed using SDS-PAGE (Supplementary Figure S1A ). Fractions containing all complex components and having a 260/280 ratio of 1.4 and higher are pulled together and concentrated to ∼30 μM and used for the cryo-EM grids preparation.
3
Fluorescent Labeling of Wadjet Complex
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A maleimide-based labeling approach was employed for fluorescent tagging of the Wadjet complex using the inserted cysteine 66 on JetA. Following the first round of size exclusion chromatography in a buffer containing 20 mM HEPES pH 7.0, 150 mM KCl, 1 mM TCEP, the JetAB subcomplex was mixed with Janelia Fluor 646 (Tocris) dye at a 1:20 molar ratio for JetAB:dye. The mixture was incubated overnight at 4°C with constant gentle rotation. On the subsequent day, excess dye was removed by passing the complex over a Superose 6 Increase 10/300 GL size exclusion column (Cytiva), and the conjugation efficiency was calculated through measurements obtained using NanoDrop (Thermo Scientific). The average labeling efficiency was determined to be ~70% for the two labeling sites in a JetA2B4 complex. The conjugated JetAB sample was then mixed with other subunits for Wadjet full complex reconstitutions (see above).
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