RNA transcribed from DNA can be divided into two groups: RNA that codes for protein and RNA that does not code for protein, or so-called non-coding RNA. Non-coding RNA can be further divided into several classes based on function. Non-coding RNAs perform a wide array of functions in living organisms, from gene regulation, to scaffolding, to catalysis. It is amazing that despite RNA having only four, chemically-similar monomers it can have such important, wide-ranging functions. Proteins which also perform wide-ranging functions in organisms have twenty common monomers that are vastly more diverse in terms of chemical or functional groups and structure. How non-coding RNA, specifically catalytic RNA or ribozymes, overcome this inherent lack of chemical and structural diversity to have impressive, intricate structures and function is the focus of this thesis. It is important to study how ribozymes are able to form intricate structure and execute function. They also have potential therapeutic applications, to control RNA viruses like HIV and oncogene transcripts, due to their ability to cleave RNA. Also, they provide a window back to a time described by the RNA World Hypothesis, a time before DNA and proteins, when RNA performed self-replication. Ribozymes overcomes its lack of diversity in monomers by being a dynamic polymer. Conformational diversity or the ability to transition from one conformation to another is critical to function of ribozymes. Nuclear magnetic resonance is a tool that is unparalleled in its ability to provide site-specific insight on time ranges from pico-seconds to thousands of seconds. The ribose dynamics of both the lead-dependent ribozyme or leadzyme and the hairpin ribozyme will be elucidated in chapters four and five, with their dynamics tied to the ribozymes’ functions. These studies represent dynamics-function assays which are essential to going beyond a static view of molecules.In this thesis, the first report of the binding kinetics of the junctionless hairpin ribozyme will be described in chapter two, which we published. The thermodynamic signature for the junctionless hairpin ribozyme will also be presented in chapter three, with important considerations of the commonly used cleavage-site modification. The kinetics and thermodynamics are essential in understanding how the junctionless hairpin ribozyme forms its active structure in a fundamental way. Lastly, a RNA-protein interaction will be discussed in chapter six. The protein is present in Trypanosoma brucei, the parasitic protozoan that causes African Sleeping Sickness in humans. The key element of the protein’s specificity for RNA was determined using in vitro selection. The specificity suggests that this protein may have a role in RNA editing. This is another case of specific interfaces being important to function.In totality, this thesis examines the structure-function paradigm prevalent in molecular biology, in a RNA-centric manner. It also goes beyond static pictures of molecules and enters into the dynamics-function realm that is essential for a more complete picture of how RNA can function as a catalyst.
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