# Dynamic association of transcription initiation proteins with chromatin at single-molecule resolution in living yeast

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2020 · $20,540

## Abstract

The life of a cell is governed by the regulated transcription of genomic information from DNA to RNA by the
enzyme RNA polymerase. Transcriptional control occurs through the actions of hundreds of proteins in the cell
nucleus whose identities and functions have been elucidated from decades of genetic and biochemical studies.
However, little is known about the timescales under which transcription-related proteins search for and target
chromatin sites in living cells, and how such dynamic associations are influenced by modifications of chromatin
architecture. This proposal aims to address these questions by applying super-resolution fluorescence
microscopy to directly visualize the diffusive movements of major transcription initiation proteins at single-
molecule resolution in budding yeast, under normal and altered states of chromatin architecture. Specifically, we
will test the hypothesis that chromatin association of transcription initiation proteins occurs with rapid kinetics in
live cells, and that chromatin remodeling and modification has a role in regulating transcription protein dynamics.
We will use live-cell single-molecule imaging to monitor the diffusive behavior of transcription initiation proteins
at high spatio-temporal resolution in the yeast nucleus. This `in vivo biochemistry' approach differs from and is
complementary to ChIP-Seq techniques that map steady-state occupancies genome-wide but provide little
information on binding dynamics. We will engineer and functionally validate DNA constructs encoding
components representative of the general transcription factors and major sequence-specific DNA binding
transcription factors fused to a self-labeling protein tag (HaloTag) that can react covalently with a cell-permeable
organic fluorophore (Janelia Fluor). Live-cell imaging of fluorescently labeled transcription factors at single-
molecule resolution measures diffusion coefficients, distinguishes between chromatin-bound and chromatin-free
populations, and estimates residence times for the bound population. Further, we will use conditional depletion
of 6 major chromatin remodelers and histone modifiers to reveal changes in the mobilities of transcription
initiation proteins and inform which among several diffusive parameters are subject to chromatin controls. This
combination of conditional mutant genetics and live-cell single-molecule imaging may transform understanding
of the kinetic mechanisms for transcription initiation and offer a new approach to other areas of yeast nuclear
and chromosome biology, including studies of DNA replication, repair, and recombination.

## Key facts

- **NIH application ID:** 10201005
- **Project number:** 3R01GM132290-02S1
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Carl Wu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $20,540
- **Award type:** 3
- **Project period:** 2020-08-01 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10201005

## Citation

> US National Institutes of Health, RePORTER application 10201005, Dynamic association of transcription initiation proteins with chromatin at single-molecule resolution in living yeast (3R01GM132290-02S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10201005. Licensed CC0.

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