1. Structure-function study of the bacteriophage 186 CII protein.
Background
- Following infection of a bacterial cell by a temperate bacteriophage, the phage can develop in one of two ways; lytic development where phage DNA is replicated and more phage particles are assembled, lysing the cell to release new viruses into environment; or lysogenic development where the phage DNA integrates into the bacterial chromosome, where it passively resides through further generations. The stable lysogenic state can quickly flipped to the alternative lytic state if the phage senses that its host is in danger. Thus this simple system serves as a tractable model for biological switches in general. Development in higher organisms can be considered as a series of nested switches, the particular state of each successive switch dictating which developmental pathway will be followed.
CII protein
- The initial "decision" made by the phage upon infection is influenced by the state of the cellular environment. The apparent master regulators of the lytic-lysogenic switch in bacteriophages lambda and 186 are the CII proteins. These proteins are unrelated to each other in sequence and share the name CII only because of the plaque morphology of cII mutants (c = clear). The CII protein of lambda has been studied extensively, and recently a crystal structure of lambda CII protein bound to DNA has been published (Datta et al., (2005) PNAS 102, 11242-11247). The CII protein of bacteriophage 186, essential for establishment of the lysogenic state, has not been so extensively characterised, but our aim is also to understand its structure-function relationship.
We know that 186 CII is a transcriptional activator, whose production shortly after infection of a host cell activates a promoter, pE, which drives expression of the CI lysogenic repressor. We predict that CII contains a helix-turn-helix DNA binding motif, and we have identified the site in the 186 genome to which it binds.
There are several questions about the function of 186 CII we would like to investigate. Ultimately we would like to determine its three dimensional structure, as we have done for the 186 CI repressor. Studying the structure-function relationship of CII will (a) help us uncover routes toward crystallisation, (b) provide data with which to check any structure that is obtained and (c) provide direct information about the mechanism of CII action. A well characterised transcriptional activator will serve as a useful biological component for the construction of larger artificial genetic circuits in the emerging field of synthetic biology.
This Honours project will involve developing a genetic screen to select for mutants of the CII protein which are unable to activate transcription. The genetic selection will consist of two parts - (i) an artificial genetic circuit has been constructed such that expression of wild type CII activates production of a gene product lethal to the host cell. Only cells expressing inactive mutants of CII will survive. (ii) To ensure that the survivors are producing activation deficient CII mutants, and not simply misfolded CII proteins, or CII mutants unable to bind DNA, you will construct an artificial promoter which is repressed, rather than activated by CII. This promoter will drive expression of a lacZ reporter. So you will select for surviving cells, where the CII protein retains the ability to bind DNA, and so repress the production of lacZ.
In addition, the project will involve identifying amino acids on RNA polymerase which are important for CII mediated activation. We will take advantage of an existing library of RNAP alpha subunit mutants for these experiments.
Other aspects of the project may involve
- using limited proteolysis of purified CII to identify the domain structure of CII,
- identifying the natural protease cleavge site within CII, a short-lived protein in vivo
- crystallisation trials. We have identified truncations of CII which are more stable than the full length protein; these may be good candidates for crystallisation trials if they can be expressed and purified in sufficient quantities.
