Thesis (M.S. in Chemistry)--Washington State University, August 2009. / Title from PDF title page (viewed on Sept. 21, 2009). "Department of Chemistry." Includes bibliographical references (p. 60-66).

Thesis (M.S.)--Bowling Green State University, 2009. / Document formatted into pages; contains 50 p. : ill. (some col.) Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: George Georgiou. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

No description available.

Molecular recognition is at the core of all biological processes whereby protein-protein interactions (PPI) relay messages to drive signaling events. However, many regulatory responses are driven by weak or transient PPI. These interactions are difficult to study using structural biology techniques because they are labile and result in heterogeneous populations. Moreover, interactions reconstituted using peptides are difficult to interpret because they lack context. In this thesis, I characterized key signaling complexes implicated in the replication checkpoint response (Dbf4-Rad53-Cdc7 complex), mitosis (Dbf4-Cdc5 complex), and DNA mismatch repair (clamp-MutL complex). I solved the crystal structures of the Dbf4-Rad53 and clamp-MutL weak complexes by generating fusions of the binding partners. The structures revealed that Dbf4 and MutL undergo subtle conformational movements upon engaging their binding partners, which were sufficient to alter both interfaces. Overall, the structures offer insight as to how Rad53 could inhibit Dbf4-Cdc7 during the replication checkpoint and how the clamp could activate MutL during mismatch repair. Acquiring the Dbf4-Cdc5 co-crystal structure required optimization the Dbf4 peptide. The Dbf4-Cdc5 and Dbf4-Rad53 complexes were relatable because both interactions were phosphorylation-independent even though Rad53 and Cdc5 are known to recognize phosphorylated targets. Dbf4 engaged a binding site on Cdc5 located opposite to the phosphoepitope binding pocket, which is reminiscent to its interaction with Rad53. Collectively, the structures of Dbf4 and its binding partners reveal that Rad53 and Cdc5 function beyond phosphoepitope recognition whereby they utilize additional binding surfaces to engage substrates. / Thesis / Doctor of Philosophy (PhD)

The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and diseases, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding RNAs, called trans-acting factors. Biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made important discoveries in characterizing how these factors regulate gene expression and determining the RNA sequence to which they bind. However, the spatiotemporal information regarding these interactions in subcellular compartments have been difficult to determine or to quantify accurately. To image and quantify native RNA and RNA–protein interactions simultaneously in situ, we developed a proximity ligation assay that combines peptide-modified RNA imaging probes. It can detect the RNAs in live cells and the interactions at a single-interaction level. Lastly, it can produce results that are easily quantifiable. We tested the specificity and sensitivity of this technique using two models: interactions between the genomic RNA and the N protein of human respiratory syncytial virus as well as those between exogenous transcripts with or without the Human antigen R (HuR) binding site and HuR. To validate this method, its accuracy and utility have been demonstrated in three models: poly(A)+ or β-actin mRNAs binding to different cytoskeleton for localization, poly(A)+ or β-actin mRNAs interacting with HuR for stabilization, and programmed cell death 4 (PDCD4) mRNA binding to HuR or T-cell intracellular antigen (TIA1) for translational regulation.

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy