All ETDs from UAB

Advisory Committee Chair

Charles L Turnbough

Advisory Committee Members

Peter J Detloff

Sunnie R Thompson

Hengbin Wang

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Transcription of ribosomal (r) DNA by RNA polymerase I (Pol I) is the initial step of ribosome synthesis. Pol I transcription is unique in its high rate of initiation, specific organization within the nucleolus and tight connection to cell growth and proliferation. Moreover, transcription elongation by Pol I is functionally coupled with rRNA processing and assembly of the ribosomes. Regulatory insights into transcription elongation by Pol I and its interface with rRNA processing are limited, despite decades of research. To fulfill that gap, we asked several important questions: Do the obvious functional divergences between Pol I and other eukaryotic polymerases extend to the catalytic core? What are the components of Pol I transcription elongation complex and how do these trans-acting factors control rRNA synthesis? What is the functional link between Pol I transcription elongation rate and ribosome biogenesis? Seeking the answers to these broad questions, we focused on specific aims: to investigate the roles of two conserved components of transcription machinery, Spt5 and the polymerase trigger loop, in yeast rRNA synthesis and ribosome assembly. Spt4/5 complex is involved in Pol I and Pol II transcription and nascent transcript maturation. In this study we demonstrated that Spt5 directly associates with Pol I and binds to the two catalytic subunits of Pol I and to the subunits A49/A34.5 that act in elongation. Interactions of Spt5 with Pol I and other components of the rDNA transcription apparatus support a direct role for Spt5 in Pol I transcription elongation. To study the degree of functional divergence between Pols I and II we examined the role of the trigger loop, a flexible domain directly involved in nucleotide addition. Analysis of Pol I trigger loop mutants suggested that analogous mutations in Pol I resulted in different phenotypes than in Pol II. The experiments using chimeric Pol II enzymes carrying the trigger loop regions of Pol I suggest that functional consequences of mutations that alter trigger loop dynamics are determined not solely by the trigger loop sequence but rather by the protein context of the enzyme. Altogether, our data suggest that Pol I and Pol II have different rate-limiting steps. In vivo characterization of the Pol I trigger loop mutants revealed ribosome assembly defects consistent with the model that the rate of elongation by Pol I is coupled to ribosome biogenesis. Based on our data we propose that optimal elongation rates are required for proper rRNA processing and that slowing Pol I elongation complexes beyond a certain rate leads to impaired ribosome biogenesis. This novel hypothesis explains the tight link between Pol I transcription elongation and rRNA maturation and highlights significance of co-transcriptional events during ribosome production. Together, these findings enhance our understanding of the mechanisms of Pol I transcription elongation and its potential roles in orchestration of rRNA processing and ribosome biogenesis.

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