Sample preparation for magnetic tweezers

Chambers were prepared as previously reported.42, 43 In brief, micro‐chambers with an approximate volume of ∼30 µL were prepared using a laser‐cut Parafilm gasket, which was heated to seal it between two coverslips (Fisherbrand, Thermo Fisher Scientific, Waltham, MA), cleaned with laboratory soap, rinsed with water, and stored in ethanol. The inlet and outlet channels were narrow to reduce evaporation of solution.44 The chamber was then incubated with 10 µg/mL purified Anti‐HA 11 Epitope tag antibody (16B12, monoclonal, Biolegend, San Diego, CA) in TXB (without magnesium glutamate) at 4˚C overnight (≤ 16 hrs) or at room temperature for 1 hr. Then, the surface was passivated with TXB (without magnesium glutamate) supplemented with 3 mg/mL α‐casein (Sigma‐Aldrich, St. Louis, MO) at room temperature for 1 h.

Stalled elongation complexes (SECs) were produced by incubating 25 nM doubly‐HA tagged E. coli RNA polymerase (Karen Adelman Laboratory, NIH), 10 nM DNA template, 50 µM GpA (initiating dinucleotide), and 10 µM ATP/UTP/GTP in TXB supplemented with 0.2 mg/mL α‐casein at 37˚C for 30 mins. SECs were then drawn into the chamber and incubated 30 min at room temperature to let the HA‐labeled RNA polymerase bind to the anti‐HA‐coated surface. The far end of the DNA from the promoter was then labeled with a 1.0 µm diameter, streptavidin‐coated paramagnetic bead (Dynabead MyOne Streptavidin T1, Invitrogen, Grand Island, NY) by incubating it with 20 µg/ml for 15 min. The experimental construct in the magnetic tweezers microscope is schematically illustrated in Supporting Information Figure S5. The extension of the DNA tether was monitored after introducing 1 mM NTPs with/without LacI (10 nM or 1 nM) in TXB supplemented with 0.2 mg/mL α‐casein.