A Family of Novel Transcriptional Regulators in the Malaria Parasite

Transcriptional regulation in the malaria parasite Plasmodium falciparum is poorly understood, both in terms of how the cis-encoded information in DNA determines gene expression and of how any trans-acting factors could arbitrate the precise temporal control of such expression. With the aim of defining a transcriptional regulatory network for P. falciparum, we have undertaken the characterization of a novel family of transcriptional regulators in the parasite. These putative transcription factors are called the Apicomplexan AP2 proteins (ApiAP2) proteins and possess DNA binding domains that are distantly related to established transcription factor domains in plant species. Prior to this work, these proteins were only annotated as hypothetical proteins that were highly conserved across the six sequenced Plasmodium genomes. These proteins represented a compelling starting-point for the dissection of transcriptional mechanisms in P. falciparum because no other family of candidate transcription factors had previously been identified.;This thesis work explores the role of these ApiAP2 proteins in P. falciparum. It begins with the initial cloning and purification of these novel Apicomplexan AP2 domains and then the demonstration that many of these domains are both necessary and sufficient to confer DNA binding specificity in vitro. We have determined the in vitro sequence preferences for every AP2 domain in P. falciparum, proving that these are bona fide DNA binding proteins with remarkable diversity in their binding specificities. We then explored the structural determinants of this DNA binding activity by crystallizing an AP2 domain along with its cognate DNA molecule. We show that the residues used to contact DNA in the Apicomplexan AP2 domain are distinct from those used in plants. Presumably, it is this divergence from plant domains that has enabled the ApiAP2 proteins to bind to a much more diverse array of DNA sequences than has been so far observed in plants. Using our ApiAP2 crystal structure, we performed an in silico docking screen to identify small molecule inhibitors that could potentially interfere with DNA binding by this domain. We have experimentally evaluated a subset of these molecules. Ultimately, we hope to define a molecular substructure that is capable of specifically and efficiently inhibiting ApiAP2 transcription factor function in vivo. No homologues of these proteins are found in the human host so they may be ideal molecular targets for drug design.