All ETDs from UAB

Advisory Committee Chair

David A Schneider

Advisory Committee Members

Mary-Ann Bjornsti

Natalia Kedishvili

Charles L Turnbough

Hengbin Wang

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Cellular growth depends on the capacity for protein synthesis, and likewise ribosome biogenesis. Therefore, robust rRNA synthesis by RNA Polymerase I (Pol I) is a critical component of cell growth. The work reported in this dissertation highlights the contribution of polymerase domains and trans-acting proteins during rRNA synthesis and ribosome biogenesis. Eukaryotes utilize three homologous nuclear RNA polymerases (RNAPs) to express their genomes. The trigger loop (TL) is a well conserved region within the active site of multi-subunit RNAPs that plays a direct role during the nucleotide addition cycle. Analysis of Pol II TL mutants has suggested that catalysis is the rate limiting step during Pol II elongation. Interestingly, the results presented here suggest that Pol I has a different rate limiting step than Pol I, perhaps translocation. This is a surprising finding given the similarity between the two enzymes and suggests that Pol I has evolved unique properties to suit its biological role. Previous studies have revealed a connection between RNAP elongation rate and RNA processing. In this work, characterization of a collection of Pol I mutants with a range of elongation rates has revealed a correlation between elongation rate and rRNA processing. These findings begin to define a role for the kinetic properties of Pol I during cotranscriptional ribosome biogenesis. Contributions from trans-acting proteins are also crucial for robust Pol I activity. We identified one such factor, Spt6, that when mutated leads to loss of Pol I from the rDNA and a striking reduction (~90%) in rRNA synthesis. However, assembly of the Pol I pre-initiation complex is not responsible for the loss of Pol I activity. Taken together these data suggest that we have uncovered the existence of a novel Pol I initiation factor and we propose that Spt6 facilitates assembly of a stable Pol I initiation complex. The work described in this dissertation spans from the Pol I active site to the influence of trans-acting proteins. These data highlight the existence of unique kinetic properties of Pol I, how these properties influence rRNA processing, and the novel finding that Pol I initiation is dependent upon Spt6.

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