All ETDs from UAB

Advisory Committee Chair

David Schneider

Advisory Committee Members

Michael Crowley

Elliot Lefkowitz

Chad Petit

William Placzek

Document Type

Dissertation

Date of Award

2019

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The production of ribosomes represents the major synthetic effort of a rapidly dividing cell, and is intimately linked to the regulation of cell growth and proliferation. Developing a greater understanding of the mechanisms that regulate ribosome biogenesis is therefore crucial to understanding cellular control of the growth cycle. Ribosome biogenesis begins with the synthesis of the 35S ribosomal RNA (rRNA) by RNA polymerase I (Pol I). This RNA is co- and post-transcriptionally processed to produce the 18S, 5.8S, and 25S rRNAs. These RNAs are critical components of ribosomes, and Pol I has been demonstrated to be a key regulation target for the ribosome biogenesis pathway. Most scholarship on the regulation of Pol I has focused on transcription initiation. More recent studies have demonstrated a link between Pol I elongation rate and efficient processing of the rRNA, indicating that Pol I transcription elongation is likely subject to regulation as well. The effect of DNA template sequence on RNA polymerase elongation has been extensively studied in prokaryotes. By contrast, the role of DNA template sequence in regulating Pol I elongation is significantly understudied. In this thesis, we have attempted to bridge this gap by studying the relationship between Pol I elongation in vitro and in vivo. First, we demonstrated that prokaryotic rho¬-independent terminator motifs induce arrest and termination in S. cerevisiae (yeast) Pol I in vitro, indicating that Pol I is sensitive to elongation-affecting DNA template sequence motifs. We next adapted Native Elongating Transcript Sequencing (NET-seq) to study Pol I occupancy in vivo. We identified reproducibly heterogeneous occupancy of Pol I throughout the 35S gene, as well as significant correlation between Pol I occupancy and the last transcribed nucleotide. Finally, we characterized occupancy in Δrpa12 yeast, a strain with altered Pol I nucleotide addition kinetics. In this mutant we observed changes in Pol I occupancy throughout the 35S gene and identified a putative third site of Pol I transcription termination. Taken together, this work simultaneously establishes DNA template sequence as a regulator of Pol I activity, and validates a technique to better explore this relationship in vivo.

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