Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Shixin Liu
Additional Thesis Advisor
Gregory M. Alushin
Abstract
Molecular machines responsible for the crucial processes of the central dogma (replication, transcription, and translation) must navigate crowded genomic tracks to robustly fulfill their crucial biological functions. Along the journey of a molecular machine, it inevitably will collide with other biological molecules competing for the same template, either static binders or additional molecular machines. While the importance of the regulation of these collisions is crucial to understanding the biological outcomes of these processes, currently no structural or atomic level details of active collisions have been acquired. This has fundamentally limited the understanding of the field to solely fitting kinetic models and inferring what happens at the collision interface instead of being able to more directly visualize and model what happens at a molecular collision. This thesis focuses on collisions with respect to one molecular machine, RNA polymerase (RNAP). RNAP performs the essential task of transcription in all living organisms, synthesizing RNAs, critical components of protein synthesis and non-coding regulatory functions in vivo, from genomic DNA template with exquisite accuracy, efficiency, and speed. RNAPs perform this task robustly and with high fidelity despite the stochasticity of the nucleotide triphosphate (NTP) binding, conformational changes, and catalysis that drive the process of transcription and the various protein barriers that are pervasive on the genome and must be overcome to generate full-length RNAs. While certain RNAP collisions can cause disastrous effects on genome stability, leading to DNA damage, mutagenesis, and aberrant gene expression, RNAP collisions are also a unique regulatory mechanism that is utilized in vivo to sense sources of damage and increase biological robustness of transcription elongation and termination. In this thesis, we use cryo-electron microscopy (cryo-EM) to visualize actively transcribing Escherichia coli (E. Coli) RNAP after collision with an inactivated restriction enzyme (EcoRI*) or another transcribing RNAP. Both collisions elicit a similar structural outcome on the RNAP. The RNAP (i) backtracks away from the site of the collision and (ii) undergoes a conformational change into a transcriptionally inactive, swiveled state. The magnitude of rotation into the swiveled swivel is coupled to DNA deformation through a previously uncharacterized structural landscape. This structural landscape implies that the swiveled status of RNAP post collision is potentially a conserved response upon RNAP collision to temporarily inactivate the enzyme until the road ahead is clear. In addition to these common outcomes, each collision scenario gave unique insights into the dynamic of that specific collision. The RNAP-EcoRI* collision structure demonstrated how an RNAP can structurally deform a roadblock protein after collision and enabled us to design points mutations to probe how the structural stability of a roadblock can impact eventual RNAP bypass. The RNAP-RNAP collision showed that the RNAP-RNAP collision is more dynamic than previously expected. The addition of an RNA hairpin into the collision system can more robustly program where these collisions occur and limit their dynamics. By resolving, to our knowledge, the first structures of actively transcribing RNAP undergoing collisions, we provide a mechanistic framework for interpreting collisions during transcription and demonstrate the feasibility of examining genomic collisions using cryo-EM.
License and Reuse Information
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Recommended Citation
Watters, John Wilson, "Structural Studies of Transcriptional Collisions" (2026). Student Theses and Dissertations. 867.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/867
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy