Barnes, Christopher
(2017)
MECHANISTIC INSIGHTS INTO EUKARYOTIC TRANSCRIPTION USING NOVEL CRYSTALLOGRAPHIC TECHNIQUES.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
DNA-directed RNA Polymerase II (Pol II) is highly conserved among eukaryotic organisms and plays a fundamental role in cellular life, specifically gene transcription. This process is universal and is at the core of gene regulation; therefore, understanding its molecular details will provide essential clues that could potentially lead to pharmacological manipulation of gene expression.
In this work we reveal, for the first time, the nature of interactions between Pol II and a complete nucleic acid scaffold. The upstream double helix lies over a wedge-shaped loop from Rpb2(862-874) that engages the minor groove, providing part of the structural framework for DNA tracking during elongation. Rudder and fork loop-1 residues insert in between the DNA/RNA hybrid and non-template strand at the upstream end of the bubble, suggesting a direct role in coordinating annealing of DNA strands. At the downstream end, interactions between the non-template strand, Rpb1(1103-1112) and Rpb2(501-510) support an open fork. These Rpb1 residues form a rigid domain with the trigger loop (TL), stabilizing its off, non-catalytic state, indicating that “on/off” conformational transitions, where the TL moves in close proximity to the matched nucleotide for catalysis, may be linked to interactions with the non-template strand.
To further examine the role, structurally, of the trigger loop and bridge helix during translocation and catalysis, we characterized the gain of function T834P variant of Pol II. Structural studies of T834P in the presence of a matched nucleotide revealed the complete TL in the on-state, a conformation only observed previously in two low resolution datasets. Our preliminary analysis suggests that kinking of the bridge helix potentially shifts equilibrium into the post-translocation state during the nucleotide addition cycle, which would prolong interactions of the trigger loop with the matched nucleotide in the insertion site. Elucidation of these structures was achieved through the use of X-ray free electron laser sources, which extended the resolution to 3.3 Å. Methodologies, like serial femtosecond crystallography and transmission electron microscopy, to aid in structural studies of highly relevant, and challenging protein targets, including multi-protein complexes of the transcriptional machinery are also discussed.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
| Creators | Email | Pitt Username | ORCID  |
|---|
| Barnes, Christopher | cob24@pitt.edu | cob24 | |
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| ETD Committee: |
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| Date: |
23 January 2017 |
| Date Type: |
Publication |
| Defense Date: |
2 December 2016 |
| Approval Date: |
23 January 2017 |
| Submission Date: |
19 December 2016 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
206 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Pharmacology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
RNA Polymerase II, Structural Biology, X-ray Free Electron Laser, Transmission Electron Microscopy, Trigger Loop, Pharmacology |
| Related URLs: |
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| Date Deposited: |
23 Jan 2017 21:02 |
| Last Modified: |
23 Jan 2018 06:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/30615 |
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