Abstract
RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states.
Highlights
RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood
An in vitro study using an immobilized promoter assay found that preinitiation complex (PIC) lacking Pol II and TFIIF remains on the promoter after initial transcription, forming a scaffold to facilitate the assembly of the reinitiation complex[6]
These findings demonstrated that Pol II can be recycled in vitro and that the specific Pol II recycling process can be isolated and studied using our in vitro two-template recycling system
Summary
RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. After the initial transcription cycle, RNA polymerases (I, II, and III) repeatedly transcribe the same gene and generate multiple RNA copies from the DNA template, contributing to robust overall transcriptional output[1,2] This transcription recycling process is best exemplified by RNA polymerase III (Pol III) recycling, which produces a large number of tRNA and 5S rRNA transcripts required for translation. While previous studies use inhibitory molecules (e.g., the detergent sarkosyl) or devised templates to separate transcription initiation from reinitiation[5], we utilize a distinct non-devised two-template approach to separate multiround transcription on the same DNA template from recycling onto the new DNA template without using detergents to block the transcription processes Among those proteins recycled with Pol II onto the new template is the human polymerase-associated factor 1 complex (PAF1C). The in vitro- and cell-based transcription recycling system we have designed opens the door for researchers to study an underappreciated but important area of transcriptional regulation: Pol II transcription recycling
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
