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.

Public Health Relevance

The proposed project aims to define fundamental mechanisms of gene regulatory biology, namely the conditions for how genes are recognized by the transcription apparatus and turned on. In the long-term, establishing general mechanisms for the transcription process defines important steps for expression of genes that either promote or prevent pathophysiological states. Knowledge of the steps of the transcription process allows directed therapeutic intervention that can improve human life.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM126704-02
Application #
9597178
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hoodbhoy, Tanya
Project Start
2018-01-01
Project End
2020-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304