# tRNA editing by deamination: Balancing affinity and specificity

> **NIH NIH R01** · OHIO STATE UNIVERSITY · 2021 · $72,298

## Abstract

Nucleic acids undergo naturally occurring chemical modifications. Over 100 different
modifications have been described and every position in the purine and pyrimidine
bases can be modified; often the sugar is also modified. Despite recent progress, the
mechanism for the biosynthesis of most modifications is not fully understood, owing,
in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas
some modifications can be efficiently formed with purified components, others may
require more intricate pathways. A model for modification interdependence, in which
one modification is a prerequisite for another, potentially explains a major hindrance
in reconstituting enzymatic activity in vitro. This model was prompted by our earlier
discovery of tRNA cytosine-to-uridine editing in eukaryotes; a reaction that had not
been recapitulated in vitro and the mechanism for which remains unknown. Formation
of m3C in vitro requires the presence of both, the T. brucei methyltransferase TRM140
and the deaminase ADAT2/3. Once formed m3C is deaminated to m3U by the same
set of enzymes.
The propose research relies heavily on our ability to determine the specific
contributions of individual residues and domains of TRM140 and ADAT2/3 to
substrate recognition and catalysis. Binding in my laboratory is determined by
Eletrophoretic Mobility Shift Assays (EMSA), while determination of catalytic activity
relies on either deamination of methylation assays based on incubation a
radioactively labeled substrate with the enzyme(s) in question. The results of both
assays are forcibly visualized and quantified by using a Typhoon-type
PhosphorImager system. The current supplement is to replace an existing unit which
has broken down and it is no longer serviceable. Without it, successful completion of
the research proposed is nearly impossible
This request is being submitted in parallel with a proposal from Dr. Kurt Fredrick in
my department at Ohio State University who also requires this equipment for his
project entitled “Molecular analysis of accurate ribosomal translocation” (R01
GM072528).

## Key facts

- **NIH application ID:** 10389330
- **Project number:** 3R01GM084065-12S1
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** Juan D Alfonzo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $72,298
- **Award type:** 3
- **Project period:** 2008-08-01 → 2023-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10389330

## Citation

> US National Institutes of Health, RePORTER application 10389330, tRNA editing by deamination: Balancing affinity and specificity (3R01GM084065-12S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10389330. Licensed CC0.

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