Circadian 24 hrs patterns of behaviour and physiology are ubiquitous in animals and plants, from bacteria to humans. Recent analyses of the molecules involved in biological rhythmicity in the fly Drosophila melanogaster and in the bread mould Neurospora crassa reveal that the key timing molecules seem to act in similar ways, suggesting that solving the problem of biological timing for one organism will serve to unravel a common molecular mechanism for all organisms. In Drosophila melanogaster, where the greatest advances have been made, two proteins PERIOD (PER) and TIMELESS (TIM), interact to generate circadian rhythmicity. They are both negative regulators of the* own transcription and they dimerise in the cytoplasm before entering the nucleus. The binding domain of PER involves a 280 amino acid region termed PAS, whereas the corresponding domain for TIM includes about 400 amino acids. Mutations which disrupt this association produce dramatic changes in periodicity. However, other than a general appreciation of the relevant dimerization domains of the two proteins, almost nothing is known about how timing information is transmitted from the cytoplasm to the nucleus.
The per gene evolves relatively quickly, and evidence from two of our laboratories suggests that the PER-TIM interaction may co-evolve, so that PER from one Dipteran species does not dimerise well to TIM from another. A comparative structure/ function approach will be used where we will initially examine the relevant sequences from the per and tim genes in a variety of Dipteran species and study their homo and heterospecific binding abilities using a yeast assay which tests for protein-protein interactions. We will simultaneously determine the molecular 'shapes' of the PER and TIM interacting surfaces in Drosophila using state-of-the-art conformational methods. The results of the conformational and yeast analyses will identify regions in the PER and TIM domains that will be critical for binding. These will be mutagenized, and tested for their binding function using both the yeast assay, and a behavioural assay of circadian behaviour. Solving the conformational structure of the two interacting domains of PER and TIM, and knowing which are the critical regions for their association may have wide applications in a number of different fields, including human health, agriculture and in the pharmaceutical industry.
Funding SchemeCSC - Cost-sharing contracts
NW7 1AA London