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Figure S1. Two step channel alignment improves resolution. Channel alignment has traditionally been performed using multispectral beads of dimensions smaller than the PSF. Stacks of 100 nm Tetraspeck bead images in separate channels were analysed using Softworx alignment software to calculate rotational, translational, tilt, and magnification offsets. When colocalising strain videos were aligned following this protocol the mean measured distance between the tagged loci was calculated to be 110 nm. Including the fine-tuning step reduced the mean error to 63 nm. Error bars are standard deviation. Figure S2. Distance verses time plots. For individual videos acquired in this study, spot separation distance in um is plotted against time in seconds. Graphs are grouped by strain as indicated. Each video is assigned an alphabetical identifier which corresponds. Figure S3. Chromatin composition at reporter loci. High-resolution ChIP profiling enrichments for the chromatin constituents indicated across the loci studied. Rpb1, PolII Ser2P, PolII Ser5P, Pcf11, Spt4, Spt5, Spt6, Spt16, and TFIIB data were generated by [90]. Histone H3 K36 monomethylation, K36 dimethylation, and K79 trimethylation data were generated by [91]. Histone H2B K123 ubiquitylation data were generated by [92]. Ino80 data were generated by [93]. Figure S4. MSD curves for operators flanking each reporter locus. Mean square displacement is plotted for the fluorescently tagged operator sequences flanking each locus used. Data for the GFP tagged lacI are shown in green and mCherry tetR in red. Data from movies taken over different time scales is combined. The grey line indicates the profile anticipated for anomalous diffusion, MSD(Δt) ∝ Δt 1/2 [89]. The green and red lines show the best-fitting lines of Brownian diffusion, MSD(Δt) ∝ Δt. Figure S5. PolII enrichment at tetO and lacO array integration sites on Chr XIV. Enrichment of PolII subunit Rpb3 along the relevant loci is shown in green [94]. Integration sites of arrays of bacterial repressor binding sites are indicated by down carats (‘V’). Locations of open reading frames, as well as their position on Watson or Crick strands, are indicated by blue and purple arrows. (A) All lacO and tetO arrays on Chr XIV were integrated between convergent genes and the terminators of the convergent genes were duplicated such that each copy flanked the insertion site. In this way all genes retained wild type copies of their terminators after insertion. (B) PolII enrichments at the integration sites of the 70.6 kb strain. TetO array was integrated between YDL089w and YDL088c, and lacO array was integrated between YDL055c and YDL054c. (C) PolII enrichments at the integration sites of the 25.3 kb strain. This strain is flanked by the ura3-1 point mutant and wild type URA3. (PDF 5372 kb)

Plasmids and strains used in this study. (PDF 460Â kb)