Fig. 3.

Correlation of Spt4/5 binding at individual DNA molecules with binding of RNApII (A and B) or Hek2 (C–E). (A) Example time records of Rpb1^SNAP549 and Spt5^DHFR-Cy5 fluorescence at four individual DNA⁴⁸⁸ locations, taken from the Rpb1^SNAP549/Spt5^DHFR-Cy5 experiment shown in Fig. 1. Colored intervals indicate times at which a fluorescent Rpb1^SNAP549 (green) and/or Spt5^DHFR-Cy5 (red) spot colocalized to the DNA⁴⁸⁸ molecule being monitored. Each Spt5^DHFR-Cy5 departure is marked according to whether it occurred before (open triangles) or simultaneously with (closed triangles) Rpb1^SNAP549 departure. Additional example records are shown in SI Appendix, Fig. S3. (B) DNA-specific binding frequencies (± SE) of Spt5^DHFR-Cy5, calculated separately for time intervals when Rpb1^SNAP549 was present or absent at the DNA. DNA-specific binding frequency is calculated as the frequency seen at DNA locations that is in excess of the background nonspecific binding seen at no-DNA locations. (C) Examples of time records of Hek2^SNAP549 and Spt5^DHFR-Cy5 fluorescence over time at three individual DNA⁴⁸⁸ locations taken from an experiment using a dual-tagged Hek2^SNAP549/Spt5^DHFR-Cy5 yeast nuclear extract in the presence of NTPs and Gal4-VP16. (D) DNA-specific binding frequencies (± SE) of Hek2^SNAP549 under transcription conditions (purple) and in negative controls (blue, orange, and yellow); see SI Appendix, Table S3. (E) Mean fraction (± SEM) of time that a DNA⁴⁸⁸ molecule had a colocalized Spt5^DHFR-Cy5, after correction for nonspecific surface binding. Separate analyses were conducted for time points at which Hek2^SNAP549 was present or absent (SI Appendix, Table S4). Data may underestimate the true occupancy of DNA locations by Spt4/5 due to possible incomplete labeling of Spt5^DHFR-Cy5, but this factor is constant across all four bars. Open bars show results of a scrambled negative control analysis of the same data (SI Appendix, Materials and Methods) showing that the correlation of Spt5 binding with Hek2 binding is not a statistical artifact. Data in D (purple bar) and E are aggregated from the experiment in C and one additional replicate.