In eukaryotes, most protein-coding genes are transcribed into messenger RNA by RNA polymerase II (Pol II). Transcription happens in three major steps: initiation, wherein Pol II recognizes and binds to the DNA upstream from a gene body; elongation, in which Pol II travels across the gene body, reads the DNA sequence, and transcribes it into RNA; and termination, in which Pol II finishes making the RNA at the end of the gene body and releases from the DNA. All three steps are exquisitely regulated; the chronic misregulation of transcription can be concomitant with a variety of disease states, underscoring the need to understand its fundamental features.

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Transcription elongation is poorly understood, but central to its intrigue is the observation that Pol II is a processive enzyme that can pause during elongation in predictable places. I contributed to a genetic screen for potential regulators of Pol II transcriptional pausing in the model yeast Saccharomyces cerevisiae. Using native elongating transcript sequencing (NET-seq), we mapped the genome-wide Pol II pausing patterns in 41 yeast strains deficient in known transcription elongation factors, chromatin remodelers, RNA processing factors, and other related proteins. These datasets provided extensive evidence to suggest that transcription elongation and pausing are thoughtfully regulated by a diverse set of factors. Furthermore, the data proved to be a rich resource for the study of both sense and antisense transcription regulation. 

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I also assisted with a project concerning the relationship between transcription elongation and phosphorylation of the C-terminal domain (CTD) of the largest subunit in budding yeast Pol II. This CTD is composed of 26 repeats of the peptide sequence Y-S-P-T-S-P-S, each of which is dynamically phosphorylated during transcription. Using quantitative mass spectroscopy, genetics, and high throughput genomics, we characterized the Pol II CTD's role as a landing pad for other regulatory complexes to perform co-transcriptional activities (e.g. splicing, chromatin remodeling) at the right place and time. We also characterized a role for the poorly-understood threonine-4 phosphoisoform in mediating the transition to transcription termination. 

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Related Publications

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Couvillon M*, Harlen KM*, Lachance KC*, Trotta KL, Smith E, Brion C, Smalec BM, Churchman LS. Transcription factor elongation is finely tuned by dozens of regulatory factors. eLife (2022).

DOI: 10.7554/eLife.78944 (eLife)

DOI: 10.1101/2021.08.15.456358 (Biorxiv)

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Harlen KH, Trotta KL, Smith EE, Mosaheb MM, Fuchs SM, Churchman LS. 

Comprehensive RNA Polymerase II Phospho-CTD Interactomes Reveal Multiple Roles for Threonine-4 in RNA Processing. Cell Reports (2016).

DOI: 10.1016/j.celrep.2016.05.010

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 (* indicates co-first authorship)

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