Projects
Here are some examples of current research projects available. For more information, please contact Keith Shearwin or Ian Dodd.
1. The role of transcriptional interference in the genetic switch of bacteriophage 186
Transcriptional Interference (TI) is the direct negative influence of transcription from one promoter on another promoter’s transcriptional activity in cis, arising from overlapping, tandem, or convergent arrangements of promoters. TI between a strong and a weak promoter tends to have little effect on the strong promoter, while producing much more significant repression of the weak promoter; thus enhancing differences in promoter strengths. TI therefore provides a means to negatively correlate the activities of two or more promoters, a potentially useful mechanism in gene regulation. This use is a possible explanation for the frequent presence in genomes of promoter arrangements likely to produce TI. See Shearwin et al. (2005) for a review of TI.
Recent work by the lab has elucidated some mechanisms of TI in prokaryotes. In collaboration with Kim Sneppen (Niels Bohr Institute, Copenhagen), mathematical and computational models of prokaryotic TI have been developed. Based upon examination of the convergent lytic and lysogenic promoters of bacteriophage 186 (located 62 bp apart), the most influential mechanisms are promoter competition, ‘sitting duck’, and RNA polymerase (RNAP) collisions. Promoter competition occurs when promoters overlap, and the occupancy of one promoter by RNAP precludes occupancy of the other promoter. ‘Sitting duck’ interference results from tandem or nearby convergent arrangements of promoters, when an elongating RNAP displaces an RNAP that has yet to begin elongation from the second promoter. RNAP collisions occur when two elongating RNAPs from convergent promoters collide, and one or both polymerases are dislodged.
Recent work in our laboratory indicates that TI between the convergent pR and pRE promoters of lambda (300 bp apart) is critical for proper setting of the lytic/lysogenic switch after infection. The lambda system involves an interesting mechanism of interference - occlusion by pausing, wherein RNAPs which initiate at pR pause over the downstream, convergent pRE promoter, interfering with the activity of pRE.
We are extending our study of TI to the corresponding promoters (pR, pE) of bacteriophage 186, using a combination of in vivo reporter assays and mathematical modeling.
2. How does the 186 anti-repressor protein flip the genetic switch from lysogeny to lytic development?
Prophage induction, or switching from the stable lysogenic state to the phage-producing lytic state involves removal of the master repressor in response to transient environmental signals, such as those that induce DNA damage. In phage lambda, this is achieved through proteolysis of the CI repressor. In bacteriophage 186 however, the same result is achieved by a specific protein-protein interaction. In response to DNA damage, 186 produces an anti-repressor protein, Tum, which inactivates the CI repressor. The aim of this project is to understand the mechanism of action of the anti-repressor.
The 186 CI protein binds cooperatively over the lytic promoter, repressing transcription of the lytic genes. It is made up of an N-terminal DNA binding domain and a C-terminal association domain. Recent X-ray crystallographic data indicates that the association domain of the repressor can form 14-mers, consisting of two heptameric rings. In vivo experiments with hybrid repressors indicate that the anti-repressor interacts not with the association domain, but rather with the N-terminal DNA binding domain.
The interaction of repressor and anti-repressor are being investigated using a variety of genetic, biochemical and molecular biological techniques. A genetic selection for isolating correctly folded, but inactive, mutants of Tum is being developed. Limited proteolysis will be used to define the domain structure of Tum. As we have available methods for purifying both proteins, surface plasmon resonance (Biacore) could be used to evaluate the interaction of anti-repressor with the wild type repressor, with mutants of the repressor and with isolated repressor domains. Other in vitro biochemical assays for protein-protein interactions will also be used. There is also the possibility of structural studies on the anti-repressor, if it proves amenable to large scale purification.
Funding Sources
Research in our laboratory is currently funded by the Human Frontiers Science Program (HFSP), the US National Institutes of Health (NIH) and the National Health and Medical Research Council (NHMRC).
