Early Elongation Control of RNA Polymerase II Transcription by TFIIS
Authors: Peck Sarah , Fox Melanie , Smith‐Kinnaman Whitney , Gao Hongyu , Liu Yunlong , Mosley Amber 
Published: November 2023

RNA polymerase II (RNAPII) transcribes multiple types of RNA, including mRNA and small nuclear RNAs (snRNAs). Transcription involves a large number of proteins that must be able to come on and off the DNA in a timely and concerted fashion. Furthermore, genes need to be expressed at the right time, place, and according to the right stimulus. Inability to properly regulate gene expression or deal with issues that occur during transcription can be catastrophic for a cell and can lead to disease and genome instability. A key event that occurs during transcription elongation is RNAPII pausing. Pausing has been implicated in the control of diverse processes including: transcriptional fidelity, elongation, RNA splicing, and termination. There are many causes of RNAPII pausing such as DNA damage, well‐positioned nucleosomes, DNA binding proteins, and so on that can cause RNAPII to stop RNA synthesis. Resolution of pausing by either resumption of elongation or termination that removes RNAPII from the DNA is necessary for proper gene expression control. We propose that there are multiple mechanisms used to deal with paused RNAPII. These mechanisms include the action of elongation factors that can keep RNAPII associated with the DNA during pausing and provide other activities that allow transcription to resume. The focus of this work is to investigate the role of the transcription elongation factor Dst1 (SII, TFIIS) in the rescue of RNAPII pausing following backtracking. Backtracking occurs when RNAPII moves backwards on the DNA template resulting in a lack of a 3′ hydroxyl in the active site, which is required for nucleotide addition and extension of the nascent RNA. Dst1 is known to stimulate the intrinsic nuclease activity of RNAPII to facilitate backtracked RNA cleavage and the resumption of RNAPII elongation. Our central hypothesis is that perturbation of DST1 will lead to the accumulation of paused RNAPII throughout the genome. In this study, we perturbed DST1 in the model organism Saccharomyces cerevisiae and measured changes in RNAPII DNA occupancy, the composition of the RNAPII interactome (+/− TFIIS), and RNAPII post translational modifications (+/− TFIIS). Our results show global changes in RNAPII occupancy at protein coding genes but not at sn/snoRNAs. These data suggest that Dst1 is required to maintain RNAPII elongation of protein coding genes. Currently we are testing the effects of Dst1 deletion on the RNAPII interactome by LC‐MS/MS. Subsequently, SAINT analysis will be performed to determine the statistical significance of the changes as a result of DST1 perturbation. The goal of this study is to broaden our understanding of regulation of transcription during RNAPII pausing. The findings of these experiments will help provide a better understanding of the resolution of RNAPII stalling during transcription elongation. Further investigations of the mechanisms that resolve paused RNAPII will not only broaden our understanding of transcription elongation and termination, but may also give us insight into RNAPII proofreading, DNA damage repair, and degradation of RNAPII.

Support or Funding Information

We would like to thank the National Science Foundation (1515748) for funding this project.