# mRNA Capping Enzyme

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2020 · $557,550

## Abstract

Project Summary. The long-term goal of this project is to understand how transcription by RNA
polymerase II (RNApII) is coupled to RNA processing and termination. This project previously developed a
model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays characteristic
phosphorylation patterns at different stages of the transcription cycle to promote binding of the appropriate
factors for co-transcriptional RNA processing. The fundamental knowledge generated by this project provides
significant insight into how the CTD phosphorylation cycle affects medically important processes such as the
stimulation of HIV transcription by the viral Tat protein and "pausing" of RNApII at developmentally regulated
genes. This project is necessary to better understand both the enzymes that mediate the changes in CTD
phosphorylation (kinases, phosphatases, etc.) as well as the proteins that recognize these patterns.
 In the next funding period, three specific aims will be pursued, with a focus on measuring dynamics of
events during transcription. The first aim continues our work directly analyzing CTD phosphorylation sites by
mass spectrometry, avoiding pitfalls associated with the monoclonal antibodies used in most CTD studies. A
modified CTD (msCTD) was engineered to discriminate individual proximal and distal repeats by mass.
Recent improvements to peptide chromatography and computational analysis will improve our accuracy and
throughput. In vivo CTD phosphorylations will be analyzed in cells where various CTD modifying enzymes are
rapidly inactivated or depleted. Analysis of RNApII associated with specific CTD binding proteins or CTD
antibodies will also be performed to determine their binding specificities. The second aim exploits our recent
discovery that CTD cycle progresses as a function of time, rather than elongation distance. This realization
allowed us to create an in vitro system that reproduces the progression of CTD phosphorylations and
associated factors on elongation complexes, facilitating real time analysis of dynamics. This immobilized
template system will be combined with the msCTD from Aim 1 to produce a high-resolution time course of CTD
phosphorylation, probing the contributions of individual kinases and phosphatases. The third aim adapts the
immobilized template assay to single-molecule microscopy. We can visualize individual transcription events
with up to three fluorescently-labeled transcription factors, providing second to millisecond time resolution of
binding kinetics. We will measure the stoichiometries, order of binding, and cooperative interactions between
multiple CTD binding and elongation complex factors. Altogether, this project will make a unique contribution to
our understanding of gene expression by providing a time-resolved picture of events that complements
inherently static techniques such as structural studies or genomics.

## Key facts

- **NIH application ID:** 9984895
- **Project number:** 5R01GM056663-22
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Stephen Buratowski
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $557,550
- **Award type:** 5
- **Project period:** 1999-07-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9984895

## Citation

> US National Institutes of Health, RePORTER application 9984895, mRNA Capping Enzyme (5R01GM056663-22). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9984895. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*