Structural analysis of the genetic switch controlling expression of the AhdI restriction-modification system

The regulation of gene expression in all organisms is dependent on complex interactions between gene-regulatory proteins and their DNA targets. A full understanding of such mechanisms requires analysis at the biophysical and biochemical level. Here, we investigate the molecular basis of the genetic switch involved in the regulation of bacterial restriction-modification systems. Restriction-modification systems in bacteria act as a form of primitive 'immune system'; they recognise foreign DNA sequences and are able to destroy invading DNA. However, expression of the gene for the enzyme that destroys the DNA must be delayed, to allow time for the host DNA to become labelled as 'self' (by a process called methylation) and thus protected from cleavage. A controller protein is responsible for this timing delay, and we have solved its molecular structure by X-ray diffraction. We have also established that the protein forms a number of complexes when it interacts with its target DNA sequence. The aim of this project is to discover the 3-dimensional structure of the various regulatory complexes bound to DNA, and to understand how they interact to allow regulation of the relevant genes. Together, these experimental approaches should provide a detailed molecular picture of the genetic switch underlying the establishment and maintenance of restriction-modification systems, many features of which should be generally applicable to a wide range of gene regulatory systems.

The regulation of gene expression is dependent on complex interactions between gene-regulatory proteins and the relevant gene-regulatory sequences. A full understanding of such mechanisms requires analysis at the biophysical and biochemical level. Here, we investigate the structural and molecular basis of the genetic switch involved in the regulation of bacterial restriction-modification systems Restriction-modification systems modulate the flow of genes among prokaryotes and play a key role in shaping bacterial evolution. However, it is vital that the expression of genes within restriction systems is subject to strict temporal control such that restriction activity is delayed with respect to methylation, otherwise auto-restriction could lead to cell death. In a wide variety of bacterial restriction-modification systems, this delay is accomplished by means of a regulator protein, the C-protein, that is required for effective transcription of its own gene, and for transcription of the endonuclease gene found on the same operon. We have recently solved the first high-resolution structure of a C-protein (C.AhdI), determined its DNA binding site and characterised the components of the switch biophysically. We subsequently established that the DNA binding site contains two operator sites, and we have defined the various complexes that can be formed, leading to a model for the mechanism of activation and repression of transcription. The current proposal seeks to build on this work by biophysical characterisation of relevant complexes, quantitative analysis of their interactions, and determination of the structure of dimeric and tetrameric complexes of C.AhdI with DNA, and of ternary complexes with the transcription activation domain of RNA polymerase. This will be supplemented by site-directed mutagenesis of key amino acid residues, and (with our collaborators) functional assays of transcription activation and repression.