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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference is a newly discovered small-RNA based immune system used by bacteria and archaea. One of the essential steps in this interference is to process long CRISPR RNAs into small CRISPR RNAs (crRNAs). Cas6 has been found to perform the CRISPR RNA processing endonuclease activity in archaea, and our goal is to study the molecular basis of Cas6's function as the endonuclease to bind and cleave the repeat RNAs. The work described in this manuscript mostly focuses on study of the structures of Cas6 protein and Cas6-RNA complexes. The main objectives of this dissertation have been outlined. In chapter 2, our goal is to understand the CRISPR processing and the enzyme involved in this small RNA generating process. A high resolution crystal structure of free Cas6 from Pyrococcus furiosus has been obtained. This structure, together with the cleavage and binding activity assays, reveals the characteristics of this endoribonuclease. In chapter 3, we want to understand the molecular mechanism of the binding and cleavage activity of Cas6, the complex structure of Cas6 and its substrate RNA was solved. Our findings suggest that Cas6 processes CRISPR RNA by a beads-on-a-string model. And Cas6 is a single-stranded RNA specific endonuclease. In chapter 4, we apply the crystallization on various repeat RNAs and Cas6 protein's non-catalytic homologue (Cas6nc) to understand how RAMP proteins, the most abundant family of proteins involved in CRISPR interference, interact with various CRISPR repeat RNAs. The structure and the complementary biochemical analysis suggest a wrap-around model of RAMP-RNA interactions, and it also sheds light on how RAMP proteins are able to specifically interact with different repeat RNA substrates.
A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Hong Li, Professor Directing Dissertation; Lloyd M. Epstein, University Representative; Brian G. Miller, Committee Member; Michael Roper, Committee Member.
Florida State University
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