Identifying an MNase Sensitive Particle associated with Rap1
Restricted (Penn State Only)
- Author:
- Pitts, Cass
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Arts
- Document Type:
- Thesis
- Thesis Supervisors:
- Lu Bai, Thesis Supervisor
Joseph C. Reese, Thesis Honors Advisor - Keywords:
- Chromatin
Nucleosome displacing factors
Rap1
SAGA
Budding yeast
Gene regulation - Abstract:
- In eukaryotes, the nucleosome is the smallest repeating unit of chromatin. It consists of 147 bp DNA wrapped around the histone octamer. The tight interactions between the histone proteins and the DNA in the nucleosome prevent the accessibility of many DNA-binding factors, including most transcription factors and RNA polymerase. A class of transcription factors in budding yeast (Saccharomyces cerevisiae), called the “nucleosome displacing factors (NDFs)”, has the unique ability to shift or evict nucleosomes near their binding sites, generate nucleosome-depleted regions, and allow transcription activators to bind and initiate transcription. One of these NDFs, Rap1, is associated with a “MNase sensitive particle;” Rap1 binding sites generate a nucleosome-size footprint when probed with low level of MNase, which can be digested when exposed to increasing concentrations of MNase. Previous research reveals that this particle does not contain a histone protein, indicating that it is not a “fragile” nucleosome but rather another protein complex with a large DNA footprint. Three potential candidates for this complex are the SAGA complex, TFIID, and the yeast Mediator. Drawing from ChIP-exo datasets, I found a significantly higher colocalization between SAGA and Rap1 than other NDFs. To test the complex in vivo, I generated strains with a subunit of the SAGA/Mediator complex knocked out. In the same strain, I also inserted an artificial sequence containing three tandem Rap1 motifs. I then performed MNase assays to assess the association of the complex with Rap1. From these anchor-away assays, TFIID and Mediator do not make up the MNase sensitive particle associated with Rap1 within the HOpr. Future anchor away assays with subunits of SAGA must be tested to experimentally validate the bioinformatic results.