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Reconstitution of Transcription Using TATA-less Promoters

使用无 TATA 启动子重建转录

基本信息

批准号：
10311016
负责人：
Jordan Taylor Feigerle
金额：
$ 1.71万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10311016
关键词：
Address Biological Assay Biology Cancer Etiology Chromatin Complex Consensus Cryoelectron Microscopy DNA DNA Polymerase II DNA-Directed RNA Polymerase Development Disease Elements Environment Eukaryota Gene Expression Generations Genes Genetic Transcription Genetic study Goals Histones Human Image In Vitro Knowledge Laboratories Lead Life Maps Measures Mediator of activation protein Methods Molecular Motivation Nucleosomes Pattern Positioning Attribute Post-Translational Protein Processing Procedures Process Proteins Proteomics Regulation Regulator Genes Reporting Repressor Proteins Research Ribosomal Proteins Saccharomyces cerevisiae Signal Transduction Site Structure Surface Plasmon Resonance TAF1 gene TATA Box TATA-Box Binding Protein Tail Testing Therapeutic Intervention Trans-Activators Transcription Initiation Transcription Initiation Site Transcription Process Transcriptional Regulation Work Yeasts experimental study improved in vivo innovation prevent promoter reconstitution response success transcription factor

项目摘要

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.

项目总结