Structural and biochemical characterization of the P-element transposase of Drosophila melanogaster

Transposons are mobile DNA segments that can change genomic location within an organism, or horizontally transfer to a new organism. They exist in all model organisms studied to date, both prokaryotic and eukaryotic, and their mobility can cause genomic rearrangements and over time, contribute to genome evolution. Transposition into a new genomic location can potentially change a protein coding segment, a regulatory transcriptional sequence, or any number of direct or indirect changes towards gene expression patterns in the cell. Although the mobile P-element is used as a molecular tool to genetically manipulate protein coding and regulatory segments in the model system Drosophila melanogaster, little mechanistic detail is known at the molecular level about the mechanism of P-element transposition. The full length P-element transposon is 2.9 kb, containing a four-exon, multi-domain protein flanked by perfect inverted repeats that are required for transposition of the element. We have taken a biochemical and structural approach towards a molecular understanding the P-element transposase mechanism; here we describe the mechanism of high-affinity site-specific DNA binding by the N-terminal THAP domain through x-ray crystallography, as well as mapping of the site-specific DNA-binding, dimerization, and GTP-binding domains onto the primary sequence of transposase through a chimeric-GFP solubility screen, for future structural studies.;Many fundamental questions remained unanswered about the mechanism of P-element transposition such as the residues responsible for specific DNA binding, GTP-binding, and catalytic functions. Previous studies in the Rio lab have discovered and initially characterized an N-terminal zinc-dependant site-specific DNA binding domain (THAP domain) in the P-element transposase, yet the mechanism of site-specific DNA-binding remained unknown. Moreover, due to recent sequencing efforts, conserved sequence elements of the P-element THAP domain have been found in over 300 animals and are thought to be evolutionarily related. This thesis describes in molecular detail the site-specific DNA-binding of the P-element transposase TRAP domain to its high affinity binding site, as well a general model for TRAP domain target site recognition. Previously thought to be a zinc-finger, this work shows that the TRAP domain targets specific DNA sites through a rare beta-sheet in the major groove interaction, with additional specificity added through a basic loop in the adjacent minor groove. Our studies suggest that THAP domains may have evolved to bind diverse sequences in the major groove through a diverse set of beta-sheet residues, yet all TRAP domains are thought to target DNA through similar modes of minor and major groove recognition using the conserved beta-sheet and basic C-terminal loop.;Additionally, a genetic screen was done on the entire P-element coding sequence to search for soluble, folded polypeptides that could be amenable to structural studeis. This work further characterizes the primary sequence and domain organization of the P-element transposase, biochemically delineating the various activities of DNA-binding, dimerization, and GTP-binding for future use in structural studies.