Single-molecule visualization assay

The surface of imaging coverslips was prepared as previously described (35), with the addition of surfaces being pegylated 5 × 24 h. For immobilization of 42 kb DNA_parS, we introduced 50 μl of ∼1 pM of 5′-biotinylated-DNA_parS molecules at a flow rate of 1 – 4 μl/min, depending on the desired end-to-end length in the experiment, in T20 buffer (40 mM Tris–HCl (pH 7.5), 20 mM NaCl, 25 nM SxG). Immediately after the flow, we further flowed 100 μl of the wash buffer (40 mM Tris–HCl, pH 7.5, 20 mM NaCl, 65 mM KCl, 25 nM SxG) at the same flow rate to ensure stretching and tethering of the other end of the DNA to the surface. By adjusting the flow, we obtained a stretch of around 15−40% of the contour length of DNA. Next, we flowed in the imaging buffer (40 mM Tris–HCl, 2 mM Trolox, 1 mM TCEP, 10 nM Catalase, 18.75 nM Glucose Oxidase, 30 mM Glucose, 2.5 mM MgCl₂, 65 mM KCl, 0.25 μg/ml BSA, 1 mM CTP/, 25 nM SxG) without ParB protein at the low flow rate (0.2 μl/min) to enable minimal disturbances to the DNA molecules before and after protein addition. Experiments were performed in the same conditions with the exception of replacing 1 mM CTP with 1 mM CTPγS where mentioned. Real-time observation of ParB diffusion was carried out by introducing ParB (5–25 nM) in the imaging buffer.

For the RNAP transcription experiments, we prepared the RNAP ternary complex as described previously by Janissen et al. (36,37). RNAP holoenzyme was stalled on the DNA_parS/T7A1 constructs at position A29 after the T7A1 promoter sequence. To do so, we added 3 nM of RNAP holoenzyme to 3 nM linear DNA_parS/T7A1 template in 20 mM Tris, 100 mM KCl, 10 mM MgCl₂, 0.05% (v/v) Tween 20 (SigmaAldrich) and 40 mg/mL BSA (New England Biolabs), pH 7.9, and incubated 10 min at 37°C. Afterwards, we added 50 μM ATP, CTP, GTP (GE Healthcare Europe), and 100 μM ApU (IBA Lifesciences GmbH) to the solution and incubated for 10 min at 30°C. To ensure that we measured the transcription dynamics of single RNAp ternary complexes, we sequestered free RNAP and RNAP that were weakly associated with the DNA by adding 100 μg/ml heparin and incubating for 10 min at 30°C. The ternary complex solution was then diluted to a final concentration of 100 pM of the RNAP:DNA_parS/T7A1 complex. The complex was flushed into the flow cell at the speed of 4 μl/min and subsequently washed for all unbound molecules in the buffer containing 40 mM Tris–HCl, 2 mM Trolox, 30 mM Glucose, 4 mM MgCl₂, 70 mM KCl, 0.25 μg/ml BSA, 1 mM CTP, 25 nM SxG. After the washing of unbound RNAP:DNA_parS/T7A1 complexes, we first subjected the buffer to the imaging buffer without ParB proteins and lacking all NTPs (40 mM Tris–HCl, 2 mM Trolox, 1 mM TCEP, 10 nM Catalase, 18.75 nM Glucose Oxidase, 30 mM Glucose, 4 mM MgCl₂, 70 mM KCl, 0.25 μg/ml BSA, 1 mM CTP, 25 nM SxG), for 3 min. This was followed by the same buffer with the addition of 25 nM ParB proteins (10 nM ParB^TMR and 15 nM WT ParB), which we incubated for 5 min to allow for DNA condensation to start prior to RNAP transcription. Following this incubation we added the final imaging buffer which allows DNA condensation and RNAP transcription (40 mM Tris–HCl, 2 mM Trolox, 1 mM TCEP, 10 nM Catalase, 18.75 nM Glucose Oxidase, 30 mM Glucose, 4 mM MgCl₂, 70 mM KCl, 0.25 μg/ml BSA, 25 nM SxG, 1 mM NTP and 2 μM GreB) and started the imaging. In these experiments the signals were obtained by alternate excitation with 100 ms exposure times for DNA-SxG (488 nm laser), ParB^TMR (561 nm laser), and RNAP^Alexa647 (647 nm laser) followed by a 2700 ms pause before the next frame. We observed 40% of the RNAP^Alexa647 molecules undergo transcription in the absence of ParB molecules (N = 18/44, Supplementary Figure S9B shows an example trace).

We used a home-built objective-TIRF microscope to achieve fluorescence imaging. We used alternating excitation of 488 nm (0.1 mW), 561 nm (12 mW) and 647 nm (12 mW) lasers in Highly Inclined and Laminated Optical sheet (HiLo) microscopy mode, to image SxG-stained DNA and TMR-labelled ParB and Alexa647-RNAP respectively. All images were acquired with an PrimeBSI sCMOS camera at an exposure time of 100 ms, with a 60× oil immersion, 1.49 NA objective (CFI APO TIRF, Nikon).