All ETDs from UAB

Advisory Committee Chair

Aaron L Lucius

Advisory Committee Members

Hui-Ting Lee

Jun Zhang

Tracy P Hamilton

Todd J Green

Document Type

Dissertation

Date of Award

2023

Abstract

Eukaryotes utilize at least three multi-subunit DNA dependent RNA polymerases (Pol I, II, and III) to synthesize RNA. The Pols are similar in structure but have distinct kinetic mechanisms that describe nucleotide addition. The product of Pol I synthesis, ribosomal RNA (rRNA) comprises the majority of the ribosome and is necessary for gene expression. Despite the importance of Pol I transcription in the cell, there is still much to understand about the roles of specific subunits in Pol I and the different factors that affect the kinetic mechanism of nucleotide addition catalyzed by Pol I. Our group has previously published the kinetic mechanisms describing the addition of one nucleotide and nine nucleotides catalyzed by Pol I. We have also published the kinetic mechanism for one nucleotide addition catalyzed by Pol I lacking the A12.2 subunit (ΔA12 Pol I). Here we extend those findings by investigating the transient state kinetics of nine nucleotide additions for ΔA12 Pol I, as well as examining the kinetic mechanism of Pol I as different nucleotides (AMP, CMP, GMP, and UMP) are incorporated into the growing RNA chain. Additionally, we examine the effect of the presence of the next templated nucleotide on the kinetic mechanism of single-nucleotide addition catalyzed by eukaryotic Pols to determine if the eukaryotic Pols contain an allosteric nucleotide binding site. Analysis of ΔA12 Pol I multi-nucleotide addition shows that the A12 subunit is responsible for fast pyrophosphate release and show that the chemical bond formation step is the rate-limiting step in the Pol I nucleotide addition cycle. In addition, our findings show that Pol I incorporates all nucleotides using the same kinetic mechanism, but that UMP is incorporated approximately twice as fast as the other three nucleotides. We also observe no evidence of an allosteric nucleotide binding site in the eukaryotic Pols, but instead we observe that the next templated nucleotide competes in the active site with the nucleotide being incorporated during the nucleotide addition cycle. Overall, this dissertation provides detailed mechanistic information about Pol I and its variants that can be used to further determine the mechanistic differences between the three eukaryotic Pols.

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