(A) The RNA-pulling assay. RNAP (green) transcribing a DNA template (blue) is attached to a bead via an avidin-biotin linkage (yellow/black). Nascent RNA (red) from the polymerase is hybridized to a 3-kb-long DNA handle (green) via a 25-base overhang; the distal end of the handle is attached via a digoxigenin-antibody linkage (black/purple) to the coverglass of a flow cell mounted on a 3D piezo stage. The optical trap (pink) holds the bead at a fixed offset from the trap center, producing a constant restoring force. The stage is moved by feedback to compensate for any elongation of the tether.
(B) The DNA-pulling assay for assisting load. The upstream end of the DNA template is attached to the coverglass surface via a digoxigenin-antibody linkage; the nascent RNA remains unbound and free to form secondary structure.
(C) 9 representative transcription records (of N = 202) from the RNA-pulling assay (red), showing elongation of the nascent RNA (in nt) vs. time. Note transcriptional pauses.
(D) 6 representative transcription records (of N = 87) from the DNA-pulling assay (blue), showing progress along the DNA (in bp) vs. time. Note transcriptional pauses.