# Reconstitution of Transcription Using TATA-less Promoters

> **NIH NIH F32** · STANFORD UNIVERSITY · 2020 · $65,310

## Abstract

Project Summary
 Gene transcription in eukaryotes is performed by RNA Polymerase II (Pol II), six general transcription
factors (GTFs) and gene specific activators, repressors and co-regulators. Misregulation of transcription and the
resulting inappropriate response to developmental, environmental, and other signals can cause disease.
Transcription begins with recognition of the promoter by the GTFs. Previous studies have focused on the
association of the TATA-box binding protein (TBP) with the TATA-box in promoters. These studies have led to
the complete assembly and structural characterization of a functional pre-initiation complex (PIC) composed of
Pol II and the GTFs including TBP on TATA-containing promoters. However, eighty percent of gene promoters
are “TATA-less”, lacking a consensus TATA sequence. TATA-less promoters require the multi-subunit TFIID
complex with thirteen to fourteen subunits in addition to TBP, rather than TBP alone. There is little, if any,
evidence of TFIID-dependent transcription of TATA-less promoters in vitro initiated at the same pattern of start
sites as in vivo. Indeed, in the yeast Saccharomyces cerevisiae, important for genetic studies, accurately initiated
TFIID-dependent transcription of TATA-less promoters in vitro has never been observed. Studies of TATA-less
gene transcription have generally been performed with naked DNA templates. Recent work has shown,
however, that genes isolated from yeast in the form of chromatin yield much higher levels of transcription, and
moreover, the patterns of transcription start sites are much more similar to those occurring in vivo. The
motivation for this proposal is the hypothesis that accurately initiated TFIID-dependent transcription of TATA-
less promoters requires a chromatin template.
 To address this hypothesis, we will isolate the S. cerevisiae TATA-less RPS5 ribosomal protein-encoding
gene in the form of chromatin assembled in vivo and transcribe with purified protein in vitro. Our first aim will be
to identify the proteins associated with the RPS5 gene in vivo by quantitative proteomics analyses of the isolated
RPS5 chromatin. We will then reconstitute transcription of RPS5 chromatin with these proteins, together with
TFIID and the many proteins shown previously to be required for transcription of TATA-containing genes in vitro.
We further aim to reconstruct a TFIID-nucleosome complex with the use of a nucleosome assembled on the
RPS5 promoter in vitro. These experiments will define the molecular requirements for association between
TFIID and a TATA-less promoter and lead to structural elucidation of a TFIID-containing PIC by cryo-electron
microscopy.

## Key facts

- **NIH application ID:** 9837453
- **Project number:** 5F32GM126704-03
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Jordan Taylor Feigerle
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $65,310
- **Award type:** 5
- **Project period:** 2018-01-01 → 2021-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9837453

## Citation

> US National Institutes of Health, RePORTER application 9837453, Reconstitution of Transcription Using TATA-less Promoters (5F32GM126704-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9837453. Licensed CC0.

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