# Structural studies of eukaryotic transcription

> **NIH NIH R01** · UNIVERSITY OF COLORADO DENVER · 2021 · $472,721

## Abstract

ABSTRACT
Cellular differentiation, development and homeostasis depend on regulation of gene expression, which is
largely focused on the DNA transcription initiation process. During transcription initiation, Mediator, a large
multi-protein complex conserved throughout eukaryotes, conveys regulatory signals to RNA polymerase II
(RNAPII), the enzyme responsible for transcription of all protein-coding genes. Depending on the specific
organism, Mediator can include 25-29 different polypeptides (total MW 1-1.5MDa) organized into Head, Middle,
Tail and CDK8 Kinase (CKM) modules, but its enzymatic activity is limited to a single Cdk8 kinase subunit.
Mounting evidence points to a mechanism largely based on conformational rearrangements that modulate
Mediator interaction with RNAPII. Consequently, a detailed understanding of Mediator structure and its
conformational dynamics is essential to elucidate how the complex regulates initiation.
Macromolecular electron microscopy (cryo-EM) is the technique of choice for characterization of large,
dynamic macromolecular assemblies. In the last couple of years, cryo-EM studies of Mediator have
dramatically advanced our molecular understanding of the complex. Here we propose cryo-EM, biochemical,
and functional studies of yeast and mammalian Mediators that build on our previous work, and that will reveal
in molecular detail the way in which critical factors modulate Mediator conformation and interaction with
RNAPII, bringing about regulation of transcription initiation.
In Aim 1 We will calculate cryo EM maps of mouse (MmMED) and human (HsMED) Mediators at near-atomic
resolution, localize metazoan-specific subunits, and determine how these subunits affect Mediator structural
rearrangements and RNAPII interaction. These results will provide an atomic-resolution understanding of
mammalian Mediator structure and reveal the structural basis for specific details of transcription
regulation by mammalian Mediator.
In Aim 2 we will use cryo-EM and biochemistry to determine the effect of CKM binding on Mediator
conformation and RNAPII association, and will investigate regulation of CKM interaction with Mediator. This will
lead to an understanding of how Mediator interaction with the CKM and concomitant structural changes
influence Mediator association with RNAPII, holoenzyme formation and, ultimately, transcription
initiation.
In Aim 3 we will use cryo-EM, image analysis and biochemistry to understand how binding of activators and
repressors to yeast and mammalian Mediators influences Mediator conformation, interaction with RNAPII and
gene expression. These studies will reveal how interaction with activators and repressors, which
generally target subunits (mostly in the Tail module) not directly involved in RNAPII interaction, can
ultimately have an effect on regulation of transcription initiation by Mediator.
Results from the studies we propose will provide a detailed understanding of mammalian Mediator structure,
and reveal...

## Key facts

- **NIH application ID:** 10236330
- **Project number:** 5R01GM067167-17
- **Recipient organization:** UNIVERSITY OF COLORADO DENVER
- **Principal Investigator:** Francisco J Asturias
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $472,721
- **Award type:** 5
- **Project period:** 2013-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10236330

## Citation

> US National Institutes of Health, RePORTER application 10236330, Structural studies of eukaryotic transcription (5R01GM067167-17). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10236330. Licensed CC0.

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