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DECIPHERING TRANSCRIPTIONAL MECHANICS ON SINGLE-MOLECULES TO THE GENOME

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2025-09-05
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Abstract

RNA polymerase (RNAP) is a powerful molecular motor that is essential for transcription - the synthesis of RNA from a DNA template. During transcription, RNAP tracks the major helical grove of DNA via a clamp downstream of its active site and this results in two interwoven consequences: (1) re-winding of downstream DNA, and (2) a relative rotation between RNAP and the DNA substrate. These events result in far reaching cellular consequences, which are experimentally challenging to investigate and thus are not yet fully understood. Here, we present two projects, each exploring one aspect of these actions. The first reveals new insights into rewinding via examination of RNAP polar dCas eviction by rewinding of the exposed RNA:DNA Hybrid. We found that dCas enzymes have an exposed R-loop only at their PAM-distal end and upon collision with RNAP, the DNA bubble is rewound, evicting the dCas gRNA. This feature was then used to design novel guide RNAs with short inverted-repeats that could resist RNAP eviction. The second project explores the use of genome-wide torsion sensors in the context of transcription in eukaryotic cells. In vivo, RNAP’s ability to rotate freely about its DNA substrate is thought to be restricted and thus its movement would result in DNA twisting as it passes through RNAP, creating torsional stress. We employed existing torsion sensors that preferentially bind either positively or negatively supercoiled DNA in yeast and evaluated their performance and limitations. From this work, we gained new insights into the complexity of sensing genome wide torsion and created a pathway to improve these tools to more precisely reveal genome wide variation in torsion.

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129 pages

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2023-08

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Wang, Michelle

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Pollack, Lois
Lambert, Guillaume

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Biophysics

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Ph. D., Biophysics

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Doctor of Philosophy

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Government Document

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dissertation or thesis

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