# Elucidating the Molecular Mechanism of Divergent Transcription

> **NIH NIH F31** · UNIVERSITY OF PENNSYLVANIA · 2024 · $48,974

## Abstract

Project Summary
The goal of this proposal is to elucidate the molecular mechanism of eukaryotic divergent transcription at
bidirectional promoters. Divergent transcription, defined as sense and antisense transcripts generated from a
common promoter, offers an additional mode of transcriptional regulation. This phenomenon has been observed
to be nearly ubiquitous and necessary for cell survival. Strand-specific sequencing methods such as global run
on sequencing and nascent elongation transcription sequencing have estimated that over 70% of active
promoters from yeast to humans exhibit divergent transcription. This is likely an underestimate as divergent
transcript pairs have been shown to be regulated and thus appear unidirectional. Additionally, of the divergent
transcript pairs, knockdown of non-coding antisense transcripts results in improper lung, heart, and brain
development. The current model for divergent transcription is that an active promoter generates a transcription
permissible state where a single Med-PIC assembles, releases a single pol II which produces a sense or
antisense transcript and then disassembles, thereby clearing space for assembly of another single PIC to repeat
the process. This model is supported by previous structural studies of Med-PIC that utilized a shortened DNA
template lacking a complete and endogenous complement of upstream activating sequences (UAS) or in the
absence of activator proteins. In my initial study, I investigated the assembly of the PIC on a natural bidirectional
promoter by utilizing a DNA template comprising an entire nucleosome-free promoter region (NFR) with UAS(s)
and flanking core promoters. Importantly, this promoter architecture is where divergent transcription typically
occurs. Interestingly, I observed that two PICs dimerize in vitro via the coactivator Mediator, hereinafter called
dMed-PIC in an activator protein dependent manner. In Aim 1, I will resolve a structurally comprehensive view
of dMed-PIC using a novel and hybrid approach of cryo-electron microscopy single particle analysis (cryo-EM
SPA) and cryo-electron tomography (cryo-ET). I will determine the assembly pathway towards divergent
transcription and thereby reveal possible regulatory mechanisms by solving the structure of dMed-PIC. In Aim
2, I will investigate transcription by dMed-PIC in vitro by yeast whole cell extract transcription assays. These
proposed experiments will use technically innovative approaches to advance our understanding of the novel
dMed-PIC and its function in divergent transcription.

## Key facts

- **NIH application ID:** 10820140
- **Project number:** 5F31GM147945-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Leon Palao
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $48,974
- **Award type:** 5
- **Project period:** 2023-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10820140

## Citation

> US National Institutes of Health, RePORTER application 10820140, Elucidating the Molecular Mechanism of Divergent Transcription (5F31GM147945-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10820140. Licensed CC0.

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