Supplementary Figure 4: N-TIR and C-TIR stabilize Pol II binding region I interaction with Pol II.
From: Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1
a, Pol II binding assay. Full-length Gdown1 and the indicated N-terminal deletion mutant fragment were incubated with Pol II and subjected to immunoprecipitation with anti-CTD antibody (8W16). Bound proteins were analyzed by immunoblot. b, EMSA with wild-type and N-terminal deletion mutant proteins. An end-labeled adenovirus ML promoter (–40 to +20) probe was incubated with 10 ng of TFIIB, 10 ng of TBP, 50 ng of TFIIF, and 50 ng of Pol II or Pol II(G). Indicated amounts of Gdown1 proteins were added to reactions. Reactions were incubated at 30 °C for 40 min and resolved by native PAGE. c, Location of Gdown1 functional domains determined by integrative modeling. C-TIR was found to exist as an overlapping region between the region (216–314) shown in dark green and the region (300–335) shown in light green. d, N-TIR location on Pol II relative to the Med2 position in the yeast Pol II–Mediator complex. A cross-link that was detected between Med2 and the RPB3 acidic region is shown in blue line.
