Supplementary Figure 8: In vitro transcription assay to characterize WT and S824-mutant TRIM28 proteins.
From: TRIM28 regulates RNA polymerase II promoter-proximal pausing and pause release
(a) TRIM28 WT, S824A, and S824D proteins were added with WT or TRIM28ΔNE in the transcription assay. Untagged WT, S824A, and S824D TRIM28 were supplied to WT or TRIM28ΔNE (0, 100 ng, and 600 ng). HSF1 was introduced after PIC assembly, immediately before adding NTPs. Transcription was allowed to proceed for 30 mins. The paused transcripts were visualized on a 12% sequencing gel. (b) The function and efficiency of TRIM28 WT, S824A, and S824D proteins to stabilize Pol II pausing in vitro. Approximately 100 ng of WT, S824A, and S824D were supplied to WT or TRIM28ΔNE during the PIC assembly on the HSPA1B template. After PIC was formed, loosely bound proteins were washed off (see Methods). HSF1 was included immediately before NTP addition. Transcription reaction was allowed for 45 minutes before visualizing nascent RNA transcripts on a 12% sequencing gel.
