Structural Characterization of Lysogeny-promoting Transcription Factors of Bacteriophage 186

Bacteriophage 186, like bacteriophage λ, is a UV-inducible temperate bacteriophage of Escherichia coli. At the DNA sequence level, these two phages show little similarity. The arrangements of promoters, genes and regulatory elements in the genome of the two phages are different, yet show similarity in the regulatory network topology. In fact, proteins with equivalent functions can be pinpointed and previous studies have shown that these proteins act as key effectors in the lytic-lysogenic decision. Studies described in this thesis were aimed at investigating these bacteriophage 186 proteins to understand their structure-function relationship. These proteins regulate transcriptional behavior and may be useful as parts for the construction of synthetic circuits. In 186, the CII protein (analogous to λ CII) is responsible for establishment of lysogeny. It is a transcriptional activator that upon binding to a pair of half sites at the 186 pE promoter, activates it to generate the initial levels of immunity repressor to allow the phage to enter lysogeny. Its unusual property of binding to half sites two DNA turns apart and its potent ability to activate pE over 400-fold, suggests it may possess a novel mechanism of promoter activation. The X-ray crystal structure of the 186 CII protein was solved. It revealed the protein adopts a tetrameric quaternary structure, consisting of rigid dimeric subunits, which further dimerize to form a tetramer. Through mass-spectrometry measurements and mutagenesis studies, it was demonstrated that this tetrameric state is necessary for its function. Molecular modelling with the crystal structure provides insight into how CII may bind DNA and interact with the host RNA polymerase to activate its promoter. The role of CII is to generate initial pools of 186 CI (analogous to λ CI). 186 CI is responsible for repressing the lytic functions of 186 to maintain lysogeny. Previous studies have shown that CI dimers multimerize into a higher order structure that interacts with DNA. High resolution structures of a CI dimer and higher order CI CTD oligomer, but no high-resolution structure of the full complex, are available. We used a combination of X-ray crystallography, small angle X-ray scattering and mass-spectrometry to demonstrate that 186 CI forms a wheel-like dodecameric structure that could act as a scaffold for DNA wrapping, giving insight into the mechanism of transcriptional regulation provided by 186 CI. Following the structural characterization of 186 CI, a temperature sensitive mutant of 186 CI was characterized to show that it can be used for temperature-based induction of gene expression. In addition, a temperature-sensitive chimeric repressor containing domains from 186 CI and λ CI was developed. This success illustrates the ability of bacteriophage regulatory proteins to be used as a source of biological parts. X-ray crystallography was extensively used in this PhD project to structurally characterize proteins. When solving novel protein structures, the generation of diffraction quality crystals and the derivatization of protein crystals with heavy atoms for experimental phasing are two commonly encountered bottlenecks. In the final section of this thesis, a technique to simultaneously optimize crystallization and derivatization of a protein is presented. Crystalline precipitate of a protein was crushed up to form a microseed stock, which was used to promote crystallization by adding it to a sparse matrix crystallization screen of the protein. An iodinated compound I3C was added to the crystallization screen, to allow incorporation of I3C into the crystal during the crystallization process. This technique was employed to solve the crystal structures of Hen-Egg White Lysozyme and a putative lysin domain Orf11 from bacteriophage P68 and should be applicable to many other crystallization targets.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202