# Structure of RNA Polymerase II

> **NIH NIH R01** · STANFORD UNIVERSITY · 2020 · $431,750

## Abstract

This grant supports our X-ray crystallographic studies of RNA polymerase II (pol II) and
associated proteins. The work began with the crystal structure determination of pol II alone at
2.8 Å and of an actively transcribing complex at 3.3 Å. There followed the structures of
transcribing complexes in both pre- and post-translocation states; structures in the post-
translocation state with nucleotides bound showing the basis for specificity (trigger loop
hypothesis); the structure of pol II in a backtracked state important for proofreading; the
structure of an initiating complex; the structure of a cocrystal of pol II with general transcription
factor TFIIB; and the structure of Mediator Head module. Specific aims for the most recent
project period were 1) extension of resolution of transcribing complex structure, 2) determination
of pre-initiation complex (PIC) structure, and 3) determination of Mediator structure, especially
the structure of a Mediator Head module-CTD complex, and extension of structural analysis to
the entire Mediator. These aims were achieved, with the completion of four very long-term
projects and publication of the results: the cryo-EM structure of a 33-protein, 1.5 megaDalton
PIC at 6-11 Å resolution; the crystal structure of a Mediator Head module-CTD complex; the
architecture of the entire Mediator by Integration Modeling of data from crystallography, cryo-EM,
chemical cross-linking, and other sources; and the cryo-EM structure of a 52-protein, 2.5
megaDalton Mediator-PIC complex at 16 Å resolution.
The goal of this project is to elucidate the mechanism of transcriptional regulation. Specific
aims for the next project period are as follows:
 1. To extend the resolution of cryo-EM of a 52-protein, 2.5 megaDalton Mediator-PIC
 complex by data collection at 300 kV on a direct electron detector, and thereby to
 address hypotheses concerning the path of the pol II CTD and Mediator-activator
interaction.
 2. To test a hypothesis for the basis of the CTD cycle, which underlies transcriptional
 regulation. We have proposed that a 79-amino acid linker preceding the 27 heptad
 repeats of the CTD is the key to the CTD cycle. We are constructing a series of pol II
 mutants, varying in the length and sequence of the linker.
 3. To elucidate the pol II transcription mechanism by time-resolved serial femtosecond
 crystallography. At present, we have structures of transcribing complexes before and
 after nucleotide addition. The proposed experiments will fill the gap between,
 revealing transcription with 10 ms resolution, well within the timescale of the process.

## Key facts

- **NIH application ID:** 10017249
- **Project number:** 5R01GM049985-27
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** JOSEPH D PUGLISI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $431,750
- **Award type:** 5
- **Project period:** 1993-08-01 → 2021-09-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10017249

## Citation

> US National Institutes of Health, RePORTER application 10017249, Structure of RNA Polymerase II (5R01GM049985-27). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/10017249. Licensed CC0.

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