# Structural Studies of RNA Polymerase II Transcription Initiation and Elongation

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $318,000

## Abstract

PROJECT SUMMARY
DNA-directed RNA Polymerase II (Pol II) is one of the most important molecules in biology. Pol II is highly-
conserved among eukaryotic organisms and plays a fundamental role in cellular life; specifically, the
transcription of genes into messenger RNA. Structural studies of Pol II have been very successful and have
allowed snapshots of Pol II in its apo form, in the process of initiation and elongation, during backtracking or
paused by DNA lesions. Moreover, single particle cryo-electron microscopy structures of Pol II in complex
with the general transcription factors (preinitiation complex), and with elongation factors have provided
views of the complexities of the initiation and elongation steps of transcription. A molecular picture of the
role that individual factors play during transcription initiation and elongation is beginning to emerge and has
generated tremendous progress towards our understanding of gene processing and regulation. Conversely,
the molecular details of nucleotide addition and the roles of conformational changes by conserved and
essential domains such as the so called “trigger loop” or “bridge helix” in substrate selection and catalysis
are not fully understood. Furthermore, pioneering studies are at lower resolution and miss critical
information to inform a complete enzymatic mechanism. This project, through extensive technology
development and innovation in structural approaches to Pol II, describes the foundation to reveal the active
site rearrangements within Pol II leading to catalysis and subsequent translocation. The studies proposed
employ a combination of state of the art technologies including time resolved X-ray crystallography, free
electron laser and single particle cryo-electron microscopy experiments to elucidate the time evolution of the
molecular events during Pol II transcriptional elongation. Importantly, orthogonal approaches are developed
to validate results from multiple independent methodologies. The Pol II system represents a model for all
cellular RNA polymerases and results obtained will represent foundational models with which Pol I, Pol III,
and prokaryotic RNA polymerases may be compared. Insight into the Pol II catalytic mechanism and
determination of how activity-altering mutants alter it will provide the framework for understanding ho
regulatory factors that target the Pol II active site might work.

## Key facts

- **NIH application ID:** 10842236
- **Project number:** 5R01GM112686-09
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Guillermo Alberto Calero
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $318,000
- **Award type:** 5
- **Project period:** 2015-01-15 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10842236

## Citation

> US National Institutes of Health, RePORTER application 10842236, Structural Studies of RNA Polymerase II Transcription Initiation and Elongation (5R01GM112686-09). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10842236. Licensed CC0.

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