Advisory Committee Chair
Aaron L Lucius
Advisory Committee Members
Richard A Dluhy
David Schneider
Charles L Turnbough Jr
Jun Zhang
Document Type
Dissertation
Date of Award
2021
Degree Name by School
Doctor of Philosophy (PhD) College of Arts and Sciences
Abstract
Eukaryotes contain at least three multi-subunit DNA dependent RNA polymerases (Pol I, II, and III). The three Pols are responsible for synthesizing specific RNAs. Pol I synthesizes the majority of the ribosomal RNA (rRNA), Pol II synthesizes all messenger RNA (mRNA) and most regulatory RNA, and Pol III synthesizes transfer RNA (tRNA) and the 5S rRNA. Despite the three Pols all synthesizing RNA, mechanistic differences are present between the polymerases. One example is the nuclease activity for each enzyme. Pol I and Pol III contain intrinsic nuclease activity conferred by specific subunits, whereas Pol II requires the association of the transcription elongation factor TFIIS. Previously, we generated kinetic mechanisms describing nucleotide addition catalyzed by Pol I and Pol I variants. Here we extended those findings by 1) performing transient-state kinetic analysis of sequential nucleotide additions catalyzed by Pol I, and 2) performing transient-state kinetic analysis of nucleotide addition catalyzed by Pol II. Interestingly, Pol I exhibited variability in the max observed rate-constant at different nucleotide incorporation positions. Additionally, a tighter nucleotide affinity was observed for GTP compared to ATP. Analysis of Pol II nucleotide addition revealed a mechanism kinetically similar to the Pol I variant Pol I ΔA12. In this dissertation we show, while similar, there are mechanistic differences between Pol I and Pol II and contribute to a broader understanding of the three Pols.
Recommended Citation
Ingram, Zachariah M., "Kinetic Mechanism Investigations of Nucleotide Addition Catalyzed by RNA Polymerase I and II" (2021). All ETDs from UAB. 621.
https://digitalcommons.library.uab.edu/etd-collection/621
