DNA-binding proteins as site-specific nucleases

Summary DNA‐binding proteins can be converted into site‐specific nucleases by linking them to the chemical nuclease 1,10‐phenanthroline‐copper. This can be readily accomplished by converting a minor groove‐proximal amino acid to a cysteine residue using site‐directed mutagenesis and then chemically modifying the sulphydryl group with 5‐iodoacetamido‐1,10‐ phenanthroline‐copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high‐resolution nuclear magnetic resonance and X‐ray crystal structures, and to locate binding sites of proteins within genomes

Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation

Co-ordinate expression of many virulence genes in the human pathogen Vibrio cholerae is under the direct control of the ToxT protein, including genes whose products are required for the biogenesis of the toxin-co-regulated pilus (TCP) and cholera toxin (CTX). This work examined interactions between ToxT and the promoters of ctx and tcpA genes. We found that a minimum of three direct repeats of the sequence TTTTGAT is required for ToxT-dependent activation of the ctx promoter, and that the region from –85 to –41 of the tcpA promoter contains elements that are responsive to ToxT-dependent activation. The role of H-NS in transcription of ctx and tcpA was also analysed. The level of activation of ctx–lacZ in an E. coli hns – strain was greatly increased even in the absence of ToxT, and was further enhanced in the presence of ToxT. In contrast, H-NS plays a lesser role in the regulation of the tcpA promoter. Electrophoretic mobility shift assays demonstrated that 6 × His-tagged ToxT directly, and specifically, interacts with both the ctx and tcpA promoters. DNase I footprinting analysis suggests that there may be two ToxT binding sites with different affinity in the ctx promoter and that ToxT binds to –84 to –41 of the tcpA promoter. In vitro transcription experiments demonstrated that ToxT alone is able to activate transcription from both promoters. We hypothesize that under conditions appropriate for ToxT-dependent gene expression, ToxT binds to AT-rich promoters that may have a specific secondary conformation, displaces H-NS and stimulates RNA polymerase resulting in transcription activation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71500/1/j.1365-2958.2002.02721.x.pd