Extended Data Figure 3: Run-off transcription under single-molecule assay conditions.

a, Isolated PICs (0.1 pmol), formed on the SNR20* short promoter fragment fused to the transcription template (covering the region −62/+636), and attached to a 2.7 kb DNA handle, was combined with increasing amounts of PICs assembled on the SNR20* short promoter, but without the handle, hereafter referred to as PIC (−62/+96). These constituents were incubated with an equal volume of a 2X NTP solution (10 ml) containing 1.6 mM ATP, 1.6 mM GTP, 1.6 mM CTP, 40 mM UTP, and 0.83 mM [α-³²P] UTP (2.5 μCi). The resulting transcripts were analyzed by gel electrophoresis. PICs fused to the DNA handle failed to support transcription alone (lane 1), but transcription activity was restored (red arrow) when a 4-fold (lane 2), 8-fold (lane 3), 12-fold (lane 4), or 15-fold (lane 5) excess of PIC (−62/+96) was added to the reactions. In lane 6, the reaction contains 1.5 pmol PIC (−62/+96). The 96-nt run-off transcription from PIC (−62/+96) is indicated (black arrow). A 25-fold excess of PIC (−62/+96) was used for single-molecule assays (Extended Figure. 2). b, 1.5 pmol aliquots of PIC (−62/+96) were introduced into different volumes of transcription buffer, such that assayed concentration of PIC varied from 37 nM to 4.5 nM. Transcription efficiency (run-off band, black) decreased with PIC concentration from ∼18% to just 2–3%. The low concentrations used in single-molecule assays (<1 nM) could not be assayed directly using gels, but we expect that the transcription efficiency is correspondingly low.