# Mechanisms of RNA Polymerase II transcription

> **NIH NIH R35** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $667,800

## Abstract

PROJECT SUMMARY
Transcription of genes in eukaryotes involves the orchestration of dozens if not hundreds of proteins enabling
each step in RNA Polymerase II (Pol II) transcription: initiation, elongation, and termination. These steps occur
dynamically on DNA templates packaged into chromatin and coincidentally with a host of cotranscriptional
events. The proposed studies broadly involve two main themes – Pol II initiation and Pol II elongation. The first
step in transcription, initiation, involves selecting a transcription start site (TSS). Promoters across eukaryotes
can have multiple TSSs and these can be used at a wide range of levels. This project will identify
comprehensively the determinants of TSS selection in the model organism Saccharomyces cerevisiae and
establish the extent to which initiation is sensitive to altered Pol II catalytic activity in other eukaryotes, an
important test of distinct mechanisms for initiation in eukaryotes. Such studies utilize massively parallel
promoter assays and genome-wide analysis of TSS usage. After initiation occurs, cotranscriptional events run
the gamut from RNA processing encompassing capping, splicing, and RNA cleavage, to modification or
remodeling of template chromatin. This project will uncover how elongation by Pol II works at a fundamental
level and probe connections between Pol II and cotranscriptional splicing; when it occurs and how it is
controlled by Pol II elongation rate. The work proposed is uniquely positioned across a wide spectrum of
biology, from fundamental mechanisms of Pol II function, to how changes to Pol II result in changes to cellular
physiology. The primary S. cerevisiae model employed has several critical advantages to exploit for these
purposes – genetics on a scale difficult to match in other systems and amenability to structural and
biochemical analyses. Gene expression mechanisms will be probed at many scales, from determining roles of
individual amino acids in the Pol II active site that are essential for proper catalytic control, to the changes in
transcriptomes caused by distinct genetic or chemical perturbations. Unique among studies on Pol II
mechanisms, the coupling of Pol II structural analyses with deep mutational scanning of the Pol II active site
will provide novel insights for how these enzymes function and evolve. The conservation of Pol II and its
regulatory machinery support the pertinence of results obtained to every eukaryotic cell, including humans
where we now recognize a complex syndrome linked to mutations in the large subunit of Pol II itself.

## Key facts

- **NIH application ID:** 10746416
- **Project number:** 5R35GM144116-03
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Craig Kaplan
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $667,800
- **Award type:** 5
- **Project period:** 2022-01-01 → 2026-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10746416

## Citation

> US National Institutes of Health, RePORTER application 10746416, Mechanisms of RNA Polymerase II transcription (5R35GM144116-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10746416. Licensed CC0.

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