Structural Insights Into Transcription Pre-Initiation Complex Assembly and Chromatin Organization Leon Aclan Palao III

Format:

Book

Thesis/Dissertation

Author/Creator:

Palao, Leon Aclan, III, author.

Contributor:

University of Pennsylvania. Biochemistry and Molecular Biophysics., degree granting institution.

Language:

English

Subjects (All):

0306.

0307.

0369.

0379.

0425.

Local Subjects:

0306.

0307.

0369.

0379.

0425.

Physical Description:

1 electronic resource (143 pages)

Contained In:

Dissertations Abstracts International 87-07B

Place of Publication:

Ann Arbor : ProQuest Dissertations and Theses, 2025

Language Note:

English

Summary:

Transcription is a ubiquitously conserved process that generates RNA from template DNA. In eukaryotes, transcription of all protein-coding genes and most non-coding RNAs is performed by RNA polymerase II (pol II). This multistep process involves chromatin modifications and rearrangement, as well as the assembly of General Transcription Factors (GTFs) and pol II on a promoter to form a Pre-initiation Complex (PIC) supported by Mediator (Med) and activator proteins. Afterwards, pol II is released and transcribes downstream of the promoter. Errors in this process can disrupt normal developmental programs and are associated with various cancers. Therefore, this process is tightly regulated through the organization and assembly of the chromatin and these transcription factors. While structural studies of transcription have been insightful, they are limited by biasing towards in vitro reconstitution conditions and away from in vivo relevance. For example, utilizing an artificial super core promoter, a shortened core promoter, or no activator proteins. To more accurately recapitulate Med-PIC formation, we employed a genuine promoter together with its cognate activator proteins. We utilized a combination of biochemistry and cryogenic electron microscopy (cryo-EM), and cryogenic electron tomography (cryo-ET) to study this process and observed a novel arrangement in which two Med-PICs dimerize through Mediator and the activator protein. Furthermore, the incorporation of additional GTFs dramatically rearranges DNA to a transcriptionally competent state. This work emphasized the value of using a more natural system and led to the development of isolating near-native specific chromatin loci aimed at understanding complex assembly and chromatin organization at these specific regions. Current methods to isolate chromatin are incompatible with cryo-EM due to low yield and unfavorable buffer conditions. Therefore, we systematically optimized each step and found the conditions crucial to generate highly pure, concentrated sample suitable for cryo-EM. This work provides a comprehensive structural understanding to Med-PIC assembly and lays the foundation for future work studying organization and assembly on near-native chromatin

Notes:

Advisors: Murakami, Kenji; Chang, Yi-Wei Committee members: Black, Ben E.; Gupta, Kushol; Gardini, Alessandro; Griesenbeck, Joachim

Source: Dissertations Abstracts International, Volume: 87-07, Section: B.

Ph.D. University of Pennsylvania 2025

Vendor supplied data

Local Notes:

School code: 0175

ISBN:

9798276005270

Access Restriction:

Restricted for use by site license