Abstract 2475 Quantifying the Repression and Induction of LacI/GalR Chimeric Proteins

The lactose repressor protein (LacI) is one of the most well studied members of the LacI/GalR family, all of which are transcriptional gene regulators. The functional unit of all LacI/GalR family members is a homodimer that includes three functional domains, the DNA binding domain, hinge linker domain, and a regulatory domain. The DNA binding domain binds to a specific sequence of DNA, allowing the dimer to block the transcriptional polymerases from transcribing genes on the operon. The hinge helix linker domain connects the DNA binding domain and the regulatory domain. The regulatory domain can change the affinity of the DNA binding domain with the presence of a ligand which differs between family members. While all members of the LacI/GalR family contain these three functional domains, LacI is unique because it has an additional C-terminal domain. This domain is also known as the tetramerization domain because it allows two LacI dimers to dimerize, overall creating a tetramer. A tetramer can bind two DNA sites, allowing the tetramer to "loop" the DNA further repressing the gene expression. Because the C-terminal domain permits looping in wild-type LacI, we hypothesize that if the C-terminal domain is added to a LacI/GalR family member then the expression of the genes on the operon will decrease because the C-terminal domain was necessary and sufficient for DNA looping. To characterize the C-terminal domain further, we decided to use previously published chimeras of the LacI/GalR family. Chimeras allow a consistent reporter allowing for testing the repression caused by the protein. These chimeras were made by taking full-length LacI and exchanging the regulatory domain with one from a different member of the LacI/GalR family. Because the C-terminal domain is only found in LacI, this regulatory domain exchange consequently led to the removal of the C-terminal domain in these chimeras. With a new focus on understanding the C-terminal domain we started to add the C-terminal domain to these chimeras. To study the C-terminal domain using these chimeras, a former student first added the LacI C-terminal domain to a chimera containing the PurR regulatory domain, creating the LacI-PurR chimera with the C-terminal domain (LacI-PurR+C). Using this LacI-PurR+C chimera, baseline repression did not significantly differ from repression compared to LacI-PurR. However, with the addition of PurR's co-repressor there was a significant increase in repression compared to LacI-PurR with its co-repressor. While overall this was not their hypothesized result, PurR has been characterized to possibly loop on its own, which might have led to the result they saw. To continue this project, we have three directions forward: (i) engineering new linker constructs between the LacI-PurR chimera and the C-terminal domain providing more flexibility for tetramerization, (ii) adding the C-terminal domain to another chimera in the family, specifically Lac- CelRc chimera that is known to not loop, and (iii) characterizing novel chimeras as candidates for C-terminal domain addition. The Olathe North SMART team, with support from the Medical Professions Academy and 3D Molecular Designs, has printed a 3-D model of tetrameric LacI bound to DNA.