Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
Browse
1 results
Search Results
Item Crenarchaeal virus-host systems : structure-function studies of crenarchaeal viruses and prokaryotic adaptive immunity(Montana State University - Bozeman, College of Letters & Science, 2011) Lintner, Nathanael Greenleaf; Chairperson, Graduate Committee: C. Martin Lawrence; Valerie Copie (co-chair)Virus-host interactions are one of the most important drivers of microbial ecology and evolution. Among the three domains of life, the least is known about viruses infecting members of the domain Archaea. This work combines a traditional hypothesis-driven molecular and biochemical approach with a structural genomics-like approach of pursuing a large number of structural targets to gain a detailed molecular understanding of a model Crenarchaeal virus-host system, Sulfolobus turreted icosahedral virus (STIV) and its Sulfolobus host. This work specifically focuses on the viral protein, A81, a putative transcriptional regulator associated with the prokaryotic adaptive immune system, CRISPR-Cas, and a large protein-RNA complex involved in CRISPR-mediated DNA interference. A81 is a protein-of-unknown-function encoded by the STIV genome. The structure of STIV-A81 reveals a unique ring-shaped octameric assembly. While structural homology-based searches fail to reveal a function for A81, the central pore has a size and charge consistent with a potential interaction with single-stranded nucleic acid. The structure of the CRIPSR-associated protein, Csa3 reveals a putative 2-domain transcription factor. The N-terminal domain is a variation on the di-nucleotide binding-domain that orchestrates dimer formation. There is a conserved 2-fold symmetric pocket on the dimer axis that likely represents a regulatory ligand-binding site implying a small-molecule regulator of CRISPR/Cas. The C-terminal domain is a winged helix-turn-helix common among transcription factors. The domain architecture of Csa3 suggests a small molecule regulator of CRISPR/Cas in the Crenarchaea. The CRISPR-associated complex for antiviral defense (CASCADE) is predicted to be central to CRISPR-mediated DNA-interference in many bacteria and Archaea. We isolated components of an archaeal CASCADE from Sulfolobus solfataricus. Csa2 expressed in S. solfataricus co-purifies with Cas5a, Cas6, Csa5 and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein, forms a stable complex with Cas5a. A recombinant Csa2-Cas5a-complex is sufficient to bind crRNA and complementary ssDNA. Transmission electron microscopy reveals an extended helical complex of variable length, perhaps due to substochiometric amounts of capping factors. Csa2 displays a crescent-shaped fold including a modified RNA-recognition motif (RRM) plus two additional domains present as insertions into the RRM. A preliminary model for this and other CASCADEs is proposed.