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We have cloned and expressed the ahdIC gene of the AhdI restriction-modification system and have purified the resulting controller (C) protein to homogeneity. The protein sequence shows a HTH motif typical of that found in many transcriptional regulators. C.AhdI is found to form a homodimer of 16.7 kDa; sedimentation equilibrium experiments show that the(More)
Restriction-modification (R-M) systems serve to protect the host bacterium from invading bacteriophage. The multi-component system includes a methyltransferase, which recognizes and methylates a specific DNA sequence, and an endonuclease which recognises the same sequence and cleaves within or close to this site. The endonuclease will only cleave DNA that(More)
The controller (C) proteins of a wide variety of restriction-modification (R-M) systems are thought to regulate expression of the endonuclease (R) gene by a genetic switch that ensures that methylation precedes endonuclease expression. Previous DNA footprinting experiments with C.AhdI have located the binding site upstream of the C and R genes in the AhdI(More)
Single crystals of purified homodimeric controller protein from Aeromonas hydrophilia (C.AhdI) have been grown under several different conditions using vapour diffusion. X-ray diffraction data have been collected using synchrotron radiation from crystals of both the native and a selenomethionine (SeMet) derivative of the protein. The native crystal form(More)
The controller protein of the Esp1396I restriction-modification (R-M) system binds differentially to three distinct operator sequences upstream of the methyltransferase (M) and endonuclease (R) genes to regulate the timing of gene expression. The crystal structure of a complex of the protein with two adjacent operator DNA sequences has been reported;(More)
Controller proteins such as C.AhdI regulate the expression of bacterial restriction-modification genes, and ensure that methylation of the host DNA precedes restriction by delaying transcription of the endonuclease. The operator DNA sequence to which C.AhdI binds consists of two adjacent binding sites, O(L) and O(R). Binding of C.AhdI to O(L) and to O(L) +(More)
Controller proteins play a key role in the temporal regulation of gene expression in bacterial restriction-modification (R-M) systems and are important mediators of horizontal gene transfer. They form the basis of a highly cooperative, concentration-dependent genetic switch involved in both activation and repression of R-M genes. Here we present(More)
Controller (C) proteins regulate the expression of restriction-modification (RM) genes in a wide variety of RM systems. However, the RM system Esp1396I is of particular interest as the C protein regulates both the restriction endonuclease (R) gene and the methyltransferase (M) gene. The mechanism of this finely tuned genetic switch depends on differential(More)
The controller protein C.Esp1396I regulates the timing of gene expression of the restriction-modification (RM) genes of the RM system Esp1396I. The molecular recognition of promoter sequences by such transcriptional regulators is poorly understood, in part because the DNA sequence motifs do not conform to a well-defined symmetry. We report here the crystal(More)
In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have(More)