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The Clustered Regularly Interspaced Short Palindromic Repeat loci found in most archaea and some bacteria contain DNA sequences (spacers) that originate from genetic invaders like viruses, transposons, and plasmids. The CRISPR clusters are transcribed into RNA and then processed into short guide CRISPR RNAs (crRNA) that are incorporated into ribonucleoprotein complexes to recognize invaders through complementary base-pairing. The Cmr complex of Pyrococcus furiosus, is an example of a ribonucleoprotein effector complex that uses crRNAs to recognize and cleave target RNA. This complex is composed of six subunits Cmr1-6 that can use diverse crRNAs of 39nt or 45nt lengths to recognize and destroy diverse target RNA sequences. The RNA cleavage activity of the Cmr complex follows a ruler mechanism by which the cleavage occurs at the 14th nucleotide form the 3' end of the crRNA in a metal dependent manner. Although the biological function of the Cmr complex is now well understood, the mechanistic details of its activity such as how the complex assembles and the roles of the different subunits, particularly the identity of the catalytic site, remain unknown. This work uses biochemical assays of RNA cleavage, in-vitro assembly studies, and structural studies to address those questions. I first addressed the role of the largest subunit of the Cmr complex Cmr2 alone and in complex with another subunit, Cmr3. Initial predictions suggested that Cmr2 may harbor the active site of the complex. However, through structural and mutagenesis studies I showed that Cmr2 does not play a direct role in the RNA-cleavage catalysis of the Cmr complex. The interaction between Cmr2 and Cmr3 results in a highly positively charged region between the two proteins that contains the nucleotide-binding site of Cmr2, as determined by solving the structure of Cmr2-Cmr3. Although Cmr3 contains multiple conserved structural elements and potentially catalytic residues, mutagenesis showed that it does not play a role in cleaving target RNAs either. To further characterize the assembly of the Cmr complex and determine the roles of its subunits, I worked in collaboration with Michael Spilman from the Scott Stagg laboratory. We obtained a low-resolution structure of the crRNA- and target RNA-bound Cmr1-6 complex. The results show that the complex has a helical architecture comprised of a Cmr2-3 foot, a Cmr4-5 twisted ladder composed of 3 Cmr4-5 steps, and a Cmr6-Cmr1 head. The crRNA-target RNA duplex binds vertically along the length of the Cmr complex. The results corroborated the previous findings on the roles of Cmr2 and Cmr3 as they showed that the two proteins are involved in specific crRNA binding. While the nature of the complex's active site remains elusive, the results provide structural support for the ruler mechanism of catalysis of the Cmr complex. The helical architecture of the complex revealed an unexpected similarity to the type I CRISPR effector complexes and suggested potential functional and structural similarities among all CRISPR effector complexes.
A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Hong Li, Professor Directing Dissertation; Joseph Travis, University Representative; Scott Stagg, Committee Member; Jamila Horabin, Committee Member; Wu-min Deng, Committee Member.
Florida State University
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