# Kinetic mechanism of transcription on native minichromosome

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2022 · $570,004

## Abstract

PROJECT SUMMARY
Advances in eukaryotic gene expression have provided a detailed and comprehensive list of transcription-related
proteins, their biochemical activities and structure-function relationships, and revealed the importance of histone
modifications and nucleosome remodeling enzymes that cooperate with sequence-specific DNA binding
transcription factors, revealing a transcriptionally poised chromatin architecture and preinitiation complex at gene
promoters and enhancers. Despite these advances, major challenges remain in the integration of chromatin
remodeling and modifying events with the complex orchestration of transcription enzymology that were
traditionally defined on naked DNA templates, and in the lack of knowledge of the timescales and kinetics by
which epigenetic and transcription proteins operate on chromatin substrates.
This proposal will address these challenges by developing an in vitro RNA Polymerase II (Pol II) transcription
system based on purified yeast minichromosomes that carry the natural set of chromatin architectures and
modifications and is responsive to transcription activators. With this minichromosome platform, we aim to
introduce defined alterations in chromatin and biochemically manipulate specific components of the transcription
apparatus to elucidate causal and temporal relationships. Extension of the analysis to the single-molecule-level
to circumvent the confounding asynchrony of ensemble reactions will allow detection of short-lived reaction
intermediates, and definition of the temporal order of intermediate events. Thus, this approach allows a direct
test of the hypothesis that the kinetics of the multi-step transcription process is regulated by chromatin-based
mechanisms, answering questions beyond the reach of current methods and synergistic with live-cell imaging of
transcription and chromatin remodeling.

## Key facts

- **NIH application ID:** 10418073
- **Project number:** 1R01GM145844-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Carl Wu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $570,004
- **Award type:** 1
- **Project period:** 2022-07-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10418073

## Citation

> US National Institutes of Health, RePORTER application 10418073, Kinetic mechanism of transcription on native minichromosome (1R01GM145844-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10418073. Licensed CC0.

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