All ETDs from UAB

Advisory Committee Chair

David A Schneider

Advisory Committee Members

Mary-Ann Bjornsti

Aaron L Lucius

Kirill Popov

Tim Townes

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The diversity of enzymes and the reactions which they catalyze is enormous. Careful study of enzymatic reaction mechanisms has taught us a great deal about chemical reactivity and has given us insight into the chemical and physical basis of life. One particularly ubiquitous class of enzymes, the RNA polymerases, are expressed in all forms of life. RNA polymerases are central components of nucleic acid metabolism and display a variety of distinctive enzymatic features; two points which solicit their thorough study. The central theme of this dissertation is the use of kinetic techniques to describe enzymatic reaction mechanisms. Although the main focus of the work is RNA polymerases, the theories and methods are broadly applicable. The introductory chapter describes the use of kinetic approaches to the study of enzyme mechanisms. In this chapter an argument is made for the use of transient – state kinetic approaches in deciphering enzyme reaction mechanisms. In the second chapter, transient – state kinetic techniques are applied to RNA Polymerase I (Pol I). Using novel experimental and computational approaches, a quantitative model describing the RNA polymerase I nucleotide incorporation mechanism was developed. This model allowed us to propose the first values of the elementary rate constants governing Pol I – catalyzed nucleotide incorporation. This is the first study of its kind in the field of Pol I and represents a significant advance in our understanding of an important enzyme. In the third chapter I present a theoretical analysis of RNA polymerase processive elongation reactions. This theoretical work lays the foundation for using ensemble kinetic data to distinguish between competing models of RNA polymerase translocation. Important insights are made regarding translocation mechanisms and experimental diagnostics are developed that will enable an investigator to distinguish between these mechanisms. In the final chapter I discuss the implications of this work on RNA polymerases and characterization of enzyme reaction mechanisms on the whole.

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