Duan, Bingbing
(2024)
Functional dissection of RNA polymerase active sites by deep mutational scanning.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Transcription in eukaryotes is carried out by three RNA polymerases (Pol), Pol I, II, and III, which are structurally conserved though they have evolved to have their own regulation and produce different classes of transcripts. At the heart of these RNA polymerases is an ultra- conserved active site domain, the trigger loop (TL), coordinating transcription speed and fidelity by critical conformational changes impacting all three steps of nucleotide addition cycle (NAC) in transcription elongation, substrate selection, catalysis, and translocation. Previous genetic and biochemical studies have shown that substitutions of TL residues disturb its balance and then alter its function. Additionally, studies from our lab have observed different types of residue-residue interactions in Pol II TL, implying the TL’s function is facilitated by residue interaction networks within and around it. Furthermore, identical mutations in a residue conserved between yeast Pol I and Pol II TLs yielded opposite biochemical phenotypes, implying even functions of conserved residues are shaped by individually evolved residue interactions in enzymatic contexts (epistasis). However, the specific mechanisms by which the TL is regulated and how it communicates with the rest of the enzyme remain unclear. Through analysis of over 15,000 alleles representing single mutants, a subset of double mutants, and evolutionarily observed TL haplotypes by deep mutational scanning, I identified intricate pairwise and higher-order epistatic interaction networks controlling TL function. Substituting residues creates allele-specific networks and propagates epistatic effects across the Pol II active site. Additionally, the interaction landscape further distinguishes alleles with similar growth phenotypes, suggesting increased resolution over the
previously reported single mutant phenotypic landscape. Furthermore, we distinguished intricated layers of higher-order epistatic interaction networks within TL haplotypes and TL residues with distinct classes of epistatic patterns in affecting these higher-order interactions. Finally, co- evolutionary analyses reveal groups of co-evolving residues across Pol II converge onto the active site, where evolutionary constraints interface with pervasive epistasis. Our studies provide a powerful system to understand the plasticity of RNA polymerase mechanism and evolution and provide the first example of pervasive epistatic landscape in a highly conserved and constrained domain within an essential enzyme.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
8 May 2024 |
| Date Type: |
Publication |
| Defense Date: |
9 January 2024 |
| Approval Date: |
8 May 2024 |
| Submission Date: |
29 March 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
204 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Pol II, Trigger loop, Genetics, Deep mutational scanning, Epistasis |
| Date Deposited: |
08 May 2024 17:43 |
| Last Modified: |
08 May 2024 17:43 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/45928 |
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