The following studies have been carried out:
Analysis of the functional geometries of bacteriophage Mu transposase and repressor:
Site directed mutagenesis of the Mu A gene:
Non sense mutations were introduced in vitro by site directed mutagenesis in regions of the transposase gene, A, allowing a more precise mapping of one subdomain of the transposase (residues 426-501) located towards the C-terminal end of the protein and involved in pA binding to the ends of the Mu genome. It was also found to be likely that the tryptophan residue at position 32 was crucial for activity.
Characterization of repressor mutants:
The differences between transposase and repressor affinities for the internal activating sequence (IAS)/operator deoxyribonucleic acid (DNA) segment, were investigated by analysis of the functional geometry of Mu repressor. The N-terminal domain of repressor was not sufficient for operator binding. Mutations in the carboxy terminal domain of Mu repressor suggested a key role for this part of the protein.
Analysis of the N-terminal domains of Mu repressor and transposase:
Repressor and transposase binding to the operator/IAS sequence suggests that, despite their having different affinities for this DNA segment, both proteins have N-terminal domains which could share similar 3-dimensional structures. In order to determine this structure by nuclear magnetic resonance (NMR), peptides were derived from repressor and transposase which would still be active for binding to the operator/IAS sequence.
Oligomerization properties of Mu repressor:
Protein crosslinking experiments where the repressor was incubated at various concentrations with a protein crosslinking reagent and analysed by electrophoresis showed extensive intermolecular crosslinking.
IHF and Mu repression;
the himA and himD genes of a phytopathogenic bacterium.
role of FIS in the maintenance of bacteriophage Mu lysogeny by the repressor.
analysis of protein protein interactions between Gin molecules during site specific recombination;
the FIS operon and its regulation;
cloning and characterization of E. coli genes regulated by FIS.
The structural and temporal architecture of protein/DNA complexes involved in two relatively well defined recombination reaction (phage Mu transposition ant the gin recombinational switch) will be investigated with particular emphasis on the role of host proteins in these processes. It is proposed to use both in vivo and in vitro approaches in these studies. The host factors include the histone-like proteins HU, IHF, and Fis (for which mutants and cloned genes are available) and an additional as yet unidentified DNA binding protein.
Funding SchemeCSC - Cost-sharing contracts