Chen et al. combined two single-molecule assays – both of which relied on 'optical tweezers' – to the determine the energy landscape for transcription. Optical tweezer experiments involve attaching a polystyrene bead (grey) to a molecule of interest, and then using a focused laser beam (red) to move the bead and thus apply a force to the molecule. (A) In the unzipping assay optical tweezers are used to pull the two strands in a DNA molecule apart. (B) A plot of force versus base pair (which is essentially distance along the DNA molecule) is jagged because it takes more energy to melt GC base pairs than AT base pairs (blue line). Nucleosomes also obstruct unzipping and increase the energy needed to melt the base pairs (red line). The grey area indicates the location of the Widom 601 positioning sequence. (C) In a transcription assay, one of the optical tweezers holds the 5' end of the DNA molecule that is being transcribed by a Pol II enzyme (green); the other optical tweezer applies a force to the enzyme itself. The mRNA molecule is the green structure to the left of the Pol II enzyme. (D) Plotting time versus base pair when a constant force is applied by the optical tweezers shows that there are small, but regular pauses in transcription (indicated by the short vertical regions in the blue line) as a consequence of the stalling sequences that were introduced in the DNA; there are no obvious pauses during transcription of the Widom 601 sequence (blue line, grey background), but there are several lengthy pauses during transcription through the nucleosome (red line, grey background). (E) By combining the rupture profile (B) and the transcription time trace (D) in a thermodynamic model that includes the geometry of the nucleosome-Pol II complex, Chen et al. were able to extract the energy landscape, which is smooth in the absence of the nucleosome (blue line), and which contains multiple peaks and valleys in the presence of the nucleosome (red line).