Fig. 1.

Detection of RNApII and Spt4/5 binding to individual surface-tethered DNA⁴⁸⁸ molecules in Rpb1^SNAP549/Spt5^DHFR-Cy5 S. cerevisiae nuclear extract. (A) Schematic of DNA⁴⁸⁸ transcription template. This DNA contains five upstream Gal4 binding sites (yellow) and the CYC1 core promoter (green) with its transcription start site (bent arrow), followed by a 300-bp cassette (pink) encoding a G-less RNA. The template has attached biotin and AF488 dye (blue star) moieties. (B) Experimental scheme. DNA⁴⁸⁸ molecules immobilized on the surface of a flow chamber (blue) were at time t = 0 incubated with yeast nuclear extract containing dye (stars)-labeled proteins Rpb1^SNAP549 and Spt5^DHFR-Cy5 along with unlabeled general transcription factors (GTFs) and other nuclear proteins. Reactions were supplemented with recombinant Gal4-VP16 activator. RNApII and Spt4/5 binding to DNA were detected as colocalization of spots of green- and red-excited fluorescence at locations of blue-excited DNA spots. (C) Images of the same microscope field of view (65 × 65 µm) in the red-, green-, and blue-excited fluorescence channels taken at various times before (blue) and after (red and green) extract addition at time t = 0. Insets show magnified views of the marked region. Absence or presence of a spot of fluorescence colocalized with a particular DNA molecule are shown by open and filled arrowheads, respectively.