# Molecular Analysis of a Yeast Transcriptional Regulator

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 2021 · $415,863

## Abstract

Project Summary/Abstract
 Gene expression is extensively controlled at the level of transcription initiation by processes that
orchestrate assembly of the multi-subunit RNA polymerase II (Pol II) preinitiation complex (PIC) at promoters.
Prevailing models derived from in vitro observations posit a defined pathway for assembly of the PIC and
formation of stable promoter-bound complexes that facilitate transcription reinitiation and perpetuate the
activated state. However, pathways for PIC assembly in vivo are not well understood, and emerging evidence
suggests much more dynamic interactions between PIC components and promoters than have been observed
in vitro. The broad, long-term objectives of the proposed project are to elucidate pathways and mechanisms
responsible for assembly and activity of PICs in vivo. To address this goal, in this proposal we will apply novel
methods that we developed in the prior period for measuring chromatin-binding dynamics in budding yeast
cells. Our multi-disciplinary approaches provide quantitative estimates of transcription factor (TF) binding
kinetics to single-copy loci including on- and off-rates as well as fractional occupancies of a DNA site by a TF
in a cell population. In Aim 1, we will measure both chromatin binding dynamics of critical transcriptional
components and RNA synthesis dynamics at a model activated gene, and mutational analyses will be used to
determine the relationships between them. In Aim 2, we will measure chromatin-binding dynamics of key
components of the PIC, genome-wide, and determine the relationships between kinetic behavior and gene
regulatory properties, RNA synthesis rates, chromatin environment, and other properties. This landscape of
kinetic properties will reveal the in vivo scope and scale of PIC assembly processes as they unfold in cells.
Our prior work indicates that the dynamic properties of a PIC component called the TATA-binding protein
(TBP) are controlled by an essential ATPase called Mot1. In Aim 3, we will use combined approaches to test
specific models for Mot1's function in gene activation, and in so doing shed light on how regulation of
chromatin binding dynamics can impact gene expression on a global scale.
 There is widespread allelic variation in TF binding sites, and such variation, as well as alterations in TF
expression levels, can lead to differences in chromatin occupancy that contribute to numerous and prevalent
human diseases, including obesity, cardiovascular disease, mental illnesses, and cancer. TFs themselves
have been largely refractory to pharmacologic intervention; instead, we propose that a quantitative
understanding of TF binding and PIC assembly dynamics in vivo will identify kinetic bottlenecks that will
provide a foundation for ultimately developing entirely new approaches to treat transcriptional defects in human
development and disease.

## Key facts

- **NIH application ID:** 10142482
- **Project number:** 5R01GM055763-21
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** David T. Auble
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $415,863
- **Award type:** 5
- **Project period:** 1997-05-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10142482

## Citation

> US National Institutes of Health, RePORTER application 10142482, Molecular Analysis of a Yeast Transcriptional Regulator (5R01GM055763-21). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10142482. Licensed CC0.

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