Journal: Science advances

Article Title: Self-assembly of promoter DNA and RNA Pol II machinery into transcriptionally active biomolecular condensates.

doi: 10.1126/sciadv.adi4565

Figure Lengend Snippet: Fig. 4. BMCs are transcriptionally active. (A) Isolated pulsed Pol II–stage BMCs are transcriptionally active. Pulsed-only in vitro transcriptions were isolated by centri- fuging at 15,000 rpm/21,000g, washed briefly in and suspended in transcription buffer. The pulsed pellet (lane 1; P) was chased for 5 min with 0.5 mM NTPs (lane 2; P/C). For comparison, lanes 3 and 4 are a complete pulse-chase assay separated into pellet (Pellet) and supernatant fractions (Sup) as in Fig. 1B. (B) Pelleted BMCs can synthesize a full-length RNA. PICs were formed with CMV promoter DNA and nuclear extract, centrifuged at 15,000 rpm/21,000g, and washed with and resuspended in transcription buffer, followed by a standard pulse-chase assay. (C) BMCs to which either BrU or BrUGAC was added were fixed and dried on coverslips and stained with anti-BrU antibody/secondary Alexa Fluor 488 antibody. A boxplot comparing the mean intensity of BrU versus BrUGAC incorporation per BMC, detected by anti-BrU and Alexa Fluor 488 secondary antibodies (n = 2) with representative microscopy images is shown. (D) Exogenously labeled RNA does not associate with the BMC. 32P- labeled RNA was isolated from a standard CMV promoter–driven pulse-chase assay and added to a second transcription reaction at the PIC formation stage. This second reaction was centrifuged; pellet and supernatant were processed, and labeled RNA was assessed as in Fig. 1B. (E) OGT inhibitor ST045849 blocks transcription when added at the PIC formation stage. A standard pulse-chase assay, control transcription (lane 1), or with ST045849 (lane 2) is shown. (F) ST045849 prevents the formation of functional BMCs when added at the PIC formation stage. Mean intensities/BMC of Alexa Fluor 488–conjugated CMV promoter DNA from one of three separate experiments are shown. The lower graph is a plot of the partition coefficient (defined as the mean sum of pixels per sample) of DNA with dimethyl sulfoxide (DMSO) or ST045849, along with representative microscopy images.

Article Snippet: For the OGT inhibition, either 0.5 μl of dimethyl sulfoxide or 0.5 μl of 10 mM ST045849 (Tocris) was added to the transcription reaction without DNA for 10 min, followed by the addition of CMV promoter DNA for 30 min.

Techniques: Isolation, In Vitro, Comparison, Pulse Chase, Staining, Microscopy, Labeling, Control, Functional Assay