# RNA modification: Mechanism and links to other metabolic pathways

> **NIH NIH R01** · SAN DIEGO STATE UNIVERSITY · 2024 · $416,431

## Abstract

Summary
 Transfer-RNAs (tRNA) are key molecules of translation, and their ability to accurately and
efficiently decode genetic information is dependent on post-transcriptional modification of
nucleotides, particularly in the critical anticodon stem loop (ASL). Deficiencies in these
modifications can be lethal, and have been linked to a variety of pleiotropic phenotypes and
human disease states. The long-term goals of this research program are to develop a detailed
understanding of the biosynthetic pathways to complex tRNA modifications, the roles these
modifications play in cellular physiology, and to identify novel targets in their pathways for
therapeutic intervention.
 This application specifically focuses on elucidating the molecular mechanisms of formation
and specificity of the universal ASL modification threonylcarbamoyladenosine (t6A) in bacteria,
and elucidating the regulatory pathways that link it to bacterial cell wall synthesis. t6A is a complex
modification found in the ASLs of tRNAs decoding ANN codons, and is critical for tRNA function
by preventing ribosomal frameshifting, promoting cognate codon recognition, facilitating tRNA
translocation, and serving as a recognition determinant for aminoacyl-tRNA synthetases.
 In the previous funding period we arrived at the first mechanistic proposal for the t6A
biosynthesis cycle in which the proteins TsaC2, TsaB and TsaD function together to install
threonylcarbamoyl on A37 of substrate tRNA, while TsaE provides an unexpected ATPase activity
required for turnover of the cycle. We also discovered that TsaE is a novel bacterial S/T/Y kinase,
and our bioinformatic analyses suggested a linkage of t6A biosynthesis to cell wall metabolism,
which raises new questions about the widely reported cell wall synthesis phenotypes associated
with t6A deficiency.
 In the current application we propose 4 specific aims that will allow us to elucidate 1) the
tRNA specificity of the bacterial t6A system, 2) the mechanism of TC-AMP transfer in t6A
biosynthesis, 3) the mechanism of ATP-hydrolysis driven turnover of the t6A cycle, and 4) the links
between TsaE and cell-wall synthesis and/or cell division. This work will be accomplished through
a combination of biochemical, structural, genetic, and physiologic approaches.

## Key facts

- **NIH application ID:** 10849650
- **Project number:** 5R01GM110588-09
- **Recipient organization:** SAN DIEGO STATE UNIVERSITY
- **Principal Investigator:** MANAL A SWAIRJO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $416,431
- **Award type:** 5
- **Project period:** 2014-09-15 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10849650

## Citation

> US National Institutes of Health, RePORTER application 10849650, RNA modification: Mechanism and links to other metabolic pathways (5R01GM110588-09). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10849650. Licensed CC0.

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